Glutamine antagonists for the treatment of cognitive deficits and psychiatric disorders

ABSTRACT

The disclosure provides compounds having formula (I): 
     
       
         
         
             
             
         
       
     
     and the pharmaceutically acceptable salts thereof, wherein R 1 , R 2 , R 2 ′, and X are as defined as set forth in the specification, for use in treating cognitive deficits and/or psychiatric disorders, such as those associated with neurological or neurodegenerative disorders, psychiatric or mood disorders, and HIV-associated neurocognitive disorders (HAND). Compounds having formula (I) are prodrugs that release glutamine analogs, e.g., 6-diazo-5-oxo-L-norleucine (DON).

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.15/884,974, filed Jan. 31, 2018, which is a continuation-in-part ofPCT/US2016/044825, filed Jul. 29, 2016, that claims the benefit of U.S.Provisional Application Nos. 62/199,317 and 62/199,566, both filed Jul.31, 2015, each of which are incorporated herein by reference in theirentirety.

FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under OD017877 ANDda037611 awarded by the National Institutes of Health. The governmenthas certain rights in the invention.

BACKGROUND

Cognitive deficits are implicated in various disorders, such asneurodegenerative, neurological, psychiatric disorders, and viraldiseases, such as human immunodeficiency virus (HIV) or acquiredimmunodeficiency syndrome (AIDS).

Parkinson's disease (PD) is a neurodegenerative condition that affectsmultiple cognitive aspects. For example, mild cognitive impairment(PD-MCI) is a comorbidity often found in PD and is associated with aprogression toward dementia (PDD) (Mak et al. Parkinsonism Relat Disord,2015. 21(8):862-70).

Multiple sclerosis (MS) is similarly fraught with cognitive deficits,such as memory impairments, particularly impairment of verbal episodicmemory (VEM) (Saenz et al. Rev Neurol (Paris), 2015. pii:S0035-3787(15)00688-8).

Mood disorders are common psychiatric illnesses that cause significantdisability and mortality worldwide. Neurocognitive changes, such asquantifiable decreases in attention, executive function, and recallmemory, have been reported in patients with mood disorders (Marvel andParadiso. Psychiatr Clin North Am. 2004; 27(1):19-viii). For example, inmajor depression, cognitive impairment can be severe and widespread, attimes meeting the definition for dementia. Similarly, the acute phase ofbipolar disorder can result in cognitive impairment that progresses to astuporous state.

Human immunodeficiency virus-1 (HIV-1) affects more than 30 millionpeople worldwide. The effects of HIV-1 worsen when it results eitherdirectly or indirectly in one or more neurological disorders.HIV-associated neurocognitive disorders (HAND) remains a prevalentdisorder and one of the central health issues in patients with chronicHIV infection on cART (Zink, J Neuroimmune Pharmacol, 2007. 2(1): p.14-9.). HAND encompasses a hierarchy of progressive neurocognitiveimpairments including asymptomatic neurocognitive impairment (ANT), mildneurocognitive disorder (MND), and severe HIV-associated dementia (HAD),the latter characterized by cognitive, motor and behavioralabnormalities (McArthur, et al., Ann. Neurol., 2010. 67(6): p. 699-714;Antinori, et al., Neurology, 2007. 69(18): p. 1789-1799; Kaul, et al.,Cell Death Differ., 2005. 12 Suppl 1: p. 878-892). In a recent studyexamining HIV-infected adults recruited from six university clinicsacross the United States, nearly half of HIV-infected individualswithout confounding conditions had some form of HAND (Heaton, et al.,Neurology, 2008. 75(23): p. 2087-96). Clinical symptoms associated withHAND include, but are not limited to, impaired short-term memory,reduced mental concentration, weakness, slowness of hand and legmovement, all of which can be accompanied by behavioral disorders, suchas depression, personality disorders, lethargy, and social withdrawal.

Broad use of cART has markedly reduced the prevalence of HIV-associateddementia (HAD) but it has had limited effect on milder forms of HAND,which now constitute the majority of new cases of HIV neurologicaldisease (Harezlak, et al., AIDS, 2011. 25(5): p. 625-633; Heaton, etal., J. Neurovirol., 2011. 17(1): p. 3-16; Robertson, et al., AIDS,2007. 21(14): p. 1915-1921). Mild HAND presents clinically as a range ofneurocognitive impairments (NCI) that do not meet the diagnosticcriteria of HAD (Antinori, et al., Neurology, 2007. 69(18): p.1789-1799) and we refer to this entity here as HIV-NCI. The biologicalbasis of HIV-NCI is largely unknown and there is currently no treatment.The impairments are often diagnosed in treated individuals with high CD4levels, sustained HIV suppression in plasma, and low or undetectablevirus in the cerebrospinal fluid (Harezlak, et al., AIDS, 2011. 25(5):p. 625-633; Simioni, et al., AIDS, 2010. 24(9): p. 1243-1250).Consistent with this mild peripheral disease profile, analyses ofautopsy brain tissues from patients who died with HIV-NCI indicateminimal pathological changes, low virus burdens, and mildly dysregulatedgene expression in the brain compared to HAD/HIVE (Borjabad, et al.,PLoS Pathog., 2011. 7(9): p. e1002213; Everall, et al., J. Neurovirol.,2009. 15(5-6): p. 360-370; Gelman, et al., PLoS One, 2012. 7(9): p.e46178). Brain imaging by ¹H-MRS suggests low-level brain inflammationand metabolic changes in neurons consistent with dysfunction rather thanneuronal apoptosis (Chang, et al., Antivir. Ther., 2003. 8(1): p. 17-26;Lentz, et al., Neurology, 2009. 72(17): p. 1465-1472; Sailasuta, et al.,PLoS One, 2012. 7(11): p. e49272). Recent metabolomic analysis ofcerebrospinal fluid from patients with mild HAND indicates a linkbetween dysregulation of energy metabolism in the brain and extent ofcognitive impairment (Dickens, et al., AIDS, 2015. 29(5):559-569). Theneuropsychological symptoms of mild HAND suggest diffusesynaptodendritic injury spanning large regions of the brain from cortexto striatum to thalamus (Ellis, et al., Nat. Rev. Neurosci., 2007. 8(1):p. 33-44); however, there is little direct documentation of such injuryas brain autopsies are rare. It is presently unclear why cART is largelyineffective in preventing or reversing HIV-NCI.

Accordingly, a need exists for methods of treating cognitive deficits,including those associated with neurodegenerative disorders,neurological diseases, psychiatric disorders, and viral disorders,including HAND, and in particular HIV-NCI.

SUMMARY

The practice of the present invention will typically employ, unlessotherwise indicated, conventional techniques of cell biology, cellculture, molecular biology, transgenic biology, microbiology,recombinant nucleic acid (e.g., DNA) technology, immunology, and RNAinterference (RNAi) which are within the skill of the art. Non-limitingdescriptions of certain of these techniques are found in the followingpublications: Ausubel, F., et al., (eds.), Current Protocols inMolecular Biology, Current Protocols in Immunology, Current Protocols inProtein Science, and Current Protocols in Cell Biology, all John Wiley &Sons, N.Y., edition as of December 2008; Sambrook, Russell, andSambrook, Molecular Cloning. A Laboratory Manual, 3^(rd) ed., ColdSpring Harbor Laboratory Press, Cold Spring Harbor, 2001; Harlow, E. andLane, D., Antibodies—A Laboratory Manual, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, 1988; Freshney, R. I., “Culture of AnimalCells, A Manual of Basic Technique”, 5th ed., John Wiley & Sons,Hoboken, N.J., 2005. Non-limiting information regarding therapeuticagents and human diseases is found in Goodman and Gilman's ThePharmacological Basis of Therapeutics, 11th Ed., McGraw Hill, 2005,Katzung, B. (ed.) Basic and Clinical Pharmacology, McGraw-Hill/Appleton& Lange 10^(th) ed. (2006) or 11th edition (July 2009). Non-limitinginformation regarding genes and genetic disorders is found in McKusick,V. A.: Mendelian Inheritance in Man. A Catalog of Human Genes andGenetic Disorders. Baltimore: Johns Hopkins University Press, 1998 (12thedition) or the more recent online database: Online MendelianInheritance in Man, OMIM™ McKusick-Nathans Institute of GeneticMedicine, Johns Hopkins University (Baltimore, Md.) and National Centerfor Biotechnology Information, National Library of Medicine (Bethesda,Md.), as of May 1, 2010, World Wide Web URL:http://www.ncbi.nlm.nih.gov/omim/ and in Online Mendelian Inheritance inAnimals (OMIA), a database of genes, inherited disorders and traits inanimal species (other than human and mouse), athttp://omia.angis.org.au/contact.shtml.

In some aspects, the presently disclosed subject matter provides the useof glutamine antagonists, such as DON, LDONV, azaserine, and acivicin,and prodrugs and analogs thereof at doses less than that used for theiranticancer efficacy to selectively abrogate cognitive deficits resultingfrom HIV infection including those defining HIV-associatedneurocognitive disorders without dementia and those included in thedefinition of HIV dementia.

Accordingly, in some aspects, the presently disclosed subject matterprovides a method for treating a subject having a cognitive deficit, themethod comprising administering to the subject at least one glutamineantagonist in an amount effective to treat the cognitive deficit.

In some aspects, the presently disclosed subject matter involves the useof at least one glutamine antagonist for treating a cognitive deficit ina subject in need thereof.

In some aspects, the presently disclosed subject matter relates to theuse of at least one glutamine antagonists for preparation of amedicament for the treatment of a cognitive deficit in a subject in needthereof.

In some aspects, the presently disclosed subject matter provides apharmaceutical composition comprising at least one glutamine antagonistin an amount effective to treat a cognitive deficit, and apharmaceutically acceptable carrier, diluent, or excipient.

In some aspects, the presently disclosed subject matter provides amethod for treating a subject having a mood or anxiety disorder, themethod comprising administering to the subject at least one glutamineantagonist in an amount effective to treat the mood or anxiety disorder.

In particular aspects, the glutamine antagonist is selected from thegroup consisting of acivicin (L-(alpha S,5S)-alpha-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid), azaserine,6-diazo-5-oxo-norleucine (DON), and 5-diazo-4-oxo-L-norvaline (L-DONV)and prodrugs or analogs thereof.

In particular aspects, the glutamine antagonist is a compounds of thedisclosure having formula (I), formula (IIA), formula (IIB), and formula(III), and the pharmaceutically acceptable salts thereof.

In more particular aspects, the cognitive deficit is due to the humanimmunodeficiency virus (HIV) infection. In such aspects, the cognitivedeficit defines HIV-associated neurocognitive disorders occurringwithout dementia, or those in association with HIV dementia.

In more particular aspects, the cognitive deficits may be due to otherdistal mechanisms than HIV infection, such as accumulation ofbeta-amyloid in patients at early stages of Alzheimer's disease orinflammation in multiple sclerosis, but are proximally mechanisticallysimilar to cognitive deficits induced by HIV.

Applicant has found that compounds of the disclosure having formula (I),formula (IIA), formula (IIB), and formula (III) are stable in plasma,liver microsomes, liver tissue, and gastrointestinal tissue, yet thesecompounds are cleaved in certain tissue cells to liberate DON. Theunexpected properties of compounds having formula (I), formula (IIA),formula (IIB), and formula (III) result in a surprising improvement intherapeutic index for treating a condition, disease, or disorder withDON and provide the maximum therapeutic benefit to a subject in need ofsuch treatment.

Applicant has also found unexpectedly that compounds of the disclosurehaving formula (I), formula (IIA), formula (IIB), and formula (III)exhibit unexpected enhanced CSF to plasma partitioning afteradministration, making them uniquely useful for the treatment acondition, disease, or disorder where central nervous system (CNS)penetration is required.

Certain aspects of the presently disclosed subject matter having beenstated hereinabove, which are addressed in whole or in part by thepresently disclosed subject matter, other aspects will become evident asthe description proceeds when taken in connection with the accompanyingExamples and Figures as best described herein below.

BRIEF DESCRIPTION OF THE FIGURES

Having thus described the presently disclosed subject matter in generalterms, reference will now be made to the accompanying Figures, which arenot necessarily drawn to scale, and wherein:

FIG. 1A, FIG. 1B, and FIG. 1C are graphs showing that prophylactictreatment of mice with DON prevents development of HIV-induced cognitiveimpairment. Six-week old C57B1 mice were treated with DON (1 mg/kg) orsaline; treatment was continued on alternate days until the end of theexperiment. 24 h after the first DON administration, groups of mice(n=8) were infected with EcoHIV (2×10⁶ pg p24/mouse) or treated withsaline by intraperitoneal inoculation. Thirty days after infection, allmice were subjected to behavioral testing in a radial arm water testmeasuring learning and memory. The data shown represent means+/−SD ofthe consecutive last 3 days of water maze testing for the indicatedgroups of mice measuring (FIG. 1A) the number of errors made by eachanimal in finding the hidden platform; (FIG. 1B) the time (latency) ittook each animal to find the hidden platform; and (FIG. 1C) the time ittook each animal to find the visible platform, as control for visual andmotor acuity of treated and untreated animals. LT1-LT4: consecutiveone-minute learning trials; RT: retention (memory) trial (the ability ofan animal to recall location of platform after a rest period). (*):p≤0.05 for pair-wise comparison by student t-test betweeninfected/untreated mice and individual other groups;

FIG. 2A, FIG. 2B, and FIG. 2C are graphs showing DON treatment of EcoHIVinfected mice with demonstrable cognitive impairment completelyabrogates cognitive disease. Mice, EcoHIV and DON doses, andexperimental procedures were as described in the DON prophylaxisexperiment shown in FIG. 1A, FIG. 1B, and FIG. 1C, except that DONadministration started 26 days after infection and was continued onalternate days until completion of behavioral testing. Water maze testswere initiated 30 days after infection. FIG. 2A shows the number oferrors made by each animal in finding the hidden platform; FIG. 2B showsthe time (latency) it took each animal to find the hidden platform; andFIG. 2C shows the time it took each animal to find the visible platform,as control for visual and motor acuity of treated and untreated animals.LT1-LT4: consecutive one-minute learning trials; RT: retention (memory)trial (the ability of an animal to recall location of platform after arest period). (*): p≤0.05 for pair-wise comparison by student t-testbetween infected/untreated mice and individual other groups;

FIG. 3A and FIG. 3B are graphs showing that DON treatment reversedHIV-mediated down-modulation of STX1A and NRGN in correlation withabrogation of cognitive defect of EcoHIV-infected mice. Relative extentof gene expression tested by QPCR in basal ganglia brain tissuesobtained from mice tested in DON treatment reversal experiment shown inFIG. 2A, FIG. 2B, and FIG. 2C. The tissues were collected 15 min afterthe last DON treatment;

FIG. 4A, FIG. 4B, and FIG. 4C are graphs showing that micromolar DONlevels were observed in plasma, CSF and basal ganglia in mice treated inthe behavioral experiments. Mouse samples were analyzed for DON usingLC-MS/MS 15 minutes post administration. DON was quantifiable in allsamples provided. Fifteen minutes after dosing, the concentration of DONaveraged from about 2.5 μM to 3 μM in plasma, and from about 0.3 μM toabout 0.5 μM in brain tissue and CSF. No significant difference wasobserved between mice infected with PBS and EcoHIV; and

FIG. 5A is a graph showing glutamate in CSF from mice treated with DON.Glutamate was quantified via LC/MS. Glutamate levels tended to be higherin EcoHIV mice versus PBS mice. In addition, DON tended to decrease theglutamate in the EcoHIV mice; however, no significant differences werefound among the treatment groups by one-way ANOVA analysis.

FIG. 5B is a graph showing that compound 25 reversed EcoHIV-inducedincreases in CSF glutamate concentration.

FIG. 6 is a graph showing a previous study demonstrated that DONadministered i.p. q.a.d at a dose of 1.6 mg/kg from the time ofimmunization attenuates EAE (Shijie, et al., 2009), but lower doses wereineffective. No other studies on DON in EAE have been reported.

FIG. 7 is a graph showing the effect of an exemplary DON prodrug on EAEdisease score when administered from the time of immunization. The DONprodrug completely prevents the development of physical signs of EAE.Significantly different from EAE+Vehicle at *P<0.05, ****P<0.0001. 2-wayANOVA treatment effect P=0.0001, n=6-8.

FIG. 8A and FIG. 8B are graphs showing the effects of an exemplary DONprodrug on EAE disease score and body weight when administered from thetime of disease onset (i.e. EAE score≥1). FIG. 8A shows the exemplaryDON prodrug significantly improves disease severity as measured by EAEdisease score. FIG. 8B shows the exemplary DON prodrug has no effect onbody weight as compared to vehicle-treated EAE mice. Significantlydifferent from EAE+Vehicle at *P<0.05, 2-way ANOVA treatment effect onEAE Disease Score P<0.05, n=7-8.

FIG. 9 is a graph showing the effects of an exemplary DON prodrug oncognition as measured by Barnes maze primary latency in EAE treatmentparadigm. EAE mice administered the exemplary DON prodrug for >8 wksfrom the time of disease onset with equivalent EAE disease scores toEAE+Vehicle mice (P<0.99) demonstrated superior long term memory withsignificantly increased primary latency deltas in the Barnes maze (Day 1Trial 1-Day 4 Trial 1). Significantly different from EAE+Vehicle at*P<0.05, n=7-8.

FIG. 10A and FIG. 10B are Barnesmaze paths and a graph, respectively,showing the effects of an exemplary DON prodrug on cognition as measuredby Barnes maze path efficiency in EAE treated mice. EAE miceadministered the exemplary DON prodrug from the time of disease onsetfor >8 weeks until equivalent EAE disease scores to EAE+Vehicle mice wasachieved (P<0.99), demonstrated improved Barnes maze performance. FIG.10A shows sample representative Barnes maze paths tracked by ANY-mazesoftware of the first trial of Day 1 or Day 4 of Barnes maze testing.FIG. 10B shows decreased path efficiency delta (Day 1 Trial 1-Day 4Trial 1) in mice treated with the exemplary DON prodrug versus vehiclesuggesting superior learning abilities in EAE+DON prodrug mice. n=7-8.

FIG. 11A and FIG. 11B are graphs showing DON prodrug (9) attenuatedLPS-induced deficits in reconsolidation memory. All mice exhibitednormal conditioning (Day 1) and reactivation (Day 2) of contextual fear.However mice that received LPS (0.3 mg/kg, i.p.) on Day 2 exhibitedreduced freezing during the reconsolidation test (Day 3) indicatingmemory impairment. *p<0.05 LPS/Vehicle vs. PBS/Vehicle.Co-administration of 9 (1.9 mg/kg, i.p.) with LPS on Day 2 attenuatedthe LPS-induced deficits in reconsolidation memory. Mice that receivedLPS and 9 were indistinguishable from PBS/Veh mice on Day 3.

FIG. 12A is a graph demonstrating different DON plasma profiles inMonkey for DON and compound 14b.

FIG. 12B is a graph showing that compound 14b exhibited enhancedCSF:plasma ratio of DON in Monkey.

FIG. 13A is a graph demonstrating different DON plasma profiles in swinefor DON, compound 14b and compound 47.

FIG. 13B is a graph showing that compounds 14b and 47 exhibited enhancedCSF delivery of DON at 60 min post-administration in swine

FIG. 13C is a graph showing that compounds 14b and 47 exhibited enhancedCSF:plasma ratio of DON at 60 min post-administration in swine.

FIG. 14A is an experimental timeline showing Chronic Social DefeatStress (CSDS) followed by chronic administration of compound 25.

FIG. 14B and FIG. 14C are graphs showing that compound 25 treatmentameliorated deficits in social behaviors induced by CSDS.

FIG. 15A, FIG. 15B, FIG. 15C, and FIG. 15D are graphs showing that DONprevented cognitive decline in the EcoHIV-infected mouse model of HAND.

FIG. 16 is a graph showing that compound 25 reversed CSDS-inducedreduction in sucrose preference test (SPT), a measure of anhedonia-likebehavior.

DETAILED DESCRIPTION

The presently disclosed subject matter now will be described more fullyhereinafter with reference to the accompanying Figures, in which some,but not all embodiments of the presently disclosed subject matter areshown. Like numbers refer to like elements throughout. The presentlydisclosed subject matter may be embodied in many different forms andshould not be construed as limited to the embodiments set forth herein;rather, these embodiments are provided so that this disclosure willsatisfy applicable legal requirements. Indeed, many modifications andother embodiments of the presently disclosed subject matter set forthherein will come to mind to one skilled in the art to which thepresently disclosed subject matter pertains having the benefit of theteachings presented in the foregoing descriptions and the associatedFigures. Therefore, it is to be understood that the presently disclosedsubject matter is not to be limited to the specific embodimentsdisclosed and that modifications and other embodiments are intended tobe included within the scope of the appended claims.

Methods of Treatment for Cognitive Deficits

Glutamine antagonists, such as 6-diazo-5-oxo-L-norleucine (DON),azaserine, and acivicin, have been shown to have anti-cancer activitiesin multiple preclinical and clinical studies. The toxicity of suchglutamine antagonists at doses necessary for their anticancer effects,however, has hampered their clinical development for cancer and otherindications. The presently disclosed subject matter provides the use ofglutamine antagonists, or prodrugs and analogs thereof, at doses lessthan that used for their anticancer efficacy, for treating cognitivedeficits, including HIV-NCI (e.g., ANI, MND, etc., as well as thoseincluded as part of the diagnostic criteria of HIV-associated dementia,also known as AIDS dementia complex. In some aspects, the presentlydisclosed subject matter provides the use of glutamine antagonists, orprodrugs and analogs thereof, at doses less than that used for theiranticancer efficacy, for treating cognitive deficits due to, orassociated with, neurodegenerative disorders, such as multiplesclerosis, Parkinson's disease, schizophrenia, Alzheimer's disease (AD),autism, or cognitive deficits due to neuroinflammation such as cerebralmalaria or encephalitis.

In one embodiment, the presently disclosed subject matter provides amethod for treating a subject having a cognitive deficit, the methodcomprising administering to the subject at least one glutamineantagonist, or prodrug or analog thereof, in an amount effective totreat the cognitive deficit.

In another embodiment, the presently disclosed subject matter involvesthe use of at least one glutamine antagonist, or prodrug or analogthereof, for treating a cognitive deficit in a subject in need thereof.

In yet other embodiments, the presently disclosed subject matterprovides a pharmaceutical composition comprising at least one glutamineantagonist, or a prodrug or analog thereof, in an amount effective totreat a cognitive deficit, and a pharmaceutically acceptable carrier,diluent, or excipient.

As used herein, a “cognitive deficit” refers to a disease, disorder, orcondition that is characterized by impairment of the mental processes ofperception, learning, memory, judgment, and/or reasoning. In someembodiments, the cognitive deficit is selected from the group consistingof dementia, and mild to moderate cognitive decline (the latterresulting in gradual incapacitation of daily activities).

As used herein, the term “dementia” refers to a terminal disease ordisorder that involves inability to think, learn, and remember such thata person's daily functioning is affected among other disabilities suchas seizures and motor detects. As used herein, the term “cognitivedecline” refers to a gradual decrease in a person's mental processes ofperception, learning, memory, judgment, and reasoning. A “mild cognitivedecline” refers to a decrease in a person's mental processes ofperception, memory, judgment, and reasoning that is less than a 40%decrease, less than a 30% decrease, less than a 20% decrease, or lessthan a 10% decrease as compared to the person's cognitive ability beforethe cognitive decline occurred.

In some embodiments, the cognitive deficit is due to a viral infection.In some embodiments, the cognitive deficit is due to the humanimmunodeficiency virus (HIV) infection. In some embodiments, the subjectis infected with HIV that is latent in T lymphocytes, other viruses thatmay be latent (e.g., JCV, Herpes) and the subject shows few or nosymptoms of the infection except cognitive deficits. In suchembodiments, the methods of treating and/or preventing may includeadministering to the subject an effective amount of an anti-viral agent.

In an aspect, the presently disclosed subject matter provides a methodfor treating a cognitive deficit in a subject due to a viral infection,the method comprising administering to the subject at least oneglutamine antagonist, or prodrug or analog thereof, in an amounteffective to treat the cognitive deficit.

In another aspect, the presently disclosed subject matter involves theuse of at least one glutamine antagonist, or prodrug or analog thereof,for treating a cognitive deficit in a subject due to a viral infection.

In yet other aspects, the presently disclosed subject matter provides apharmaceutical composition comprising at least one glutamine antagonist,or a prodrug or analog thereof, in an amount effective to treat acognitive deficit due to a viral infection in a subject, and apharmaceutically acceptable carrier, diluent, or excipient.

In an aspect, the presently disclosed subject matter provides a methodof preventing a cognitive deficit in a subject having a viral infection,the method comprising administering to the subject at least oneglutamine antagonist, or prodrug or analog thereof, in an amounteffective to prevent the cognitive deficit.

In another aspect, the presently disclosed subject matter involves theuse of at least one glutamine antagonist, or prodrug or analog thereof,for preventing a cognitive deficit in a subject having a viralinfection.

In yet other aspects, the presently disclosed subject matter provides apharmaceutical composition comprising at least one glutamine antagonist,or a prodrug or analog thereof, in an amount effective to prevent acognitive deficit in a subject having a viral infection, and apharmaceutically acceptable carrier, diluent, or excipient.

In an aspect, the presently disclosed subject matter provides a methodfor treating HIV-induced cognitive impairment in a subject in needthereof, the method comprising administering to the subject at least oneglutamine antagonist, or prodrug or analog thereof, in an amounteffective to treat the HIV-induced cognitive impairment in the subject.

In another aspect, the presently disclosed subject matter involves theuse of at least one glutamine antagonist, or prodrug or analog thereof,for treating HIV-induced cognitive impairment in a subject in needthereof.

In yet other aspects, the presently disclosed subject matter provides apharmaceutical composition comprising at least one glutamine antagonist,or a prodrug or analog thereof, in an amount effective to treatHIV-induced cognitive impairment in a subject in need thereof, and apharmaceutically acceptable carrier, diluent, or excipient.

In an aspect, the presently disclosed subject matter provides a methodof preventing HIV-induced cognitive impairment in a subject in needthereof, the method comprising administering to the subject at least oneglutamine antagonist, or prodrug or analog thereof, in an amounteffective to prevent the cognitive deficit.

In another aspect, the presently disclosed subject matter involves theuse of at least one glutamine antagonist, or prodrug or analog thereof,for preventing HIV-induced cognitive impairment in a subject in needthereof.

In yet other aspects, the presently disclosed subject matter provides apharmaceutical composition comprising at least one glutamine antagonist,or a prodrug or analog thereof, in an amount effective to preventHIV-induced cognitive impairment in a subject in need thereof, and apharmaceutically acceptable carrier, diluent, or excipient.

In some embodiments, the cognitive deficit is part of but not completeclinical definition HIV-associated dementia, also known as AIDS dementiacomplex. In some embodiments, the cognitive deficit is AC. In someembodiments, the cognitive deficit is MND. In some embodiments, thecognitive deficit is HIV-NCI.

Generally, HIV can affect any part of the nervous system, such as thebrain, spinal cord, or peripheral nerves. Depending on which part of thenervous system is involved, symptoms of neurologic disease inHIV-infected individuals may include memory loss, headache, dizziness,weakness, numbness, pain, vision changes, or trouble walking.

It should be appreciated that some neurologic disorders, such asopportunistic infections, primary central nervous system lymphoma(PCNSL), and HIV-associated dementia (HAD) occur only in patients withadvanced AIDS. Other neurologic conditions, however, such asHIV-associated neuropathy and HIV-associated asymptomatic neurocognitiveimpairment (ANI), and HIV-associated mild neurocognitive disorder (MND),occur even in patients with well-controlled HIV.

More particularly, neurologic conditions associated with HIV include,but are not limited to, cerebral toxoplasmosis, cryptococcal meningitis,cytomegalovirus (CMV) encephalitis, distal symmetric polyneuropathy(DSP), HIV-associated dementia (HAD), HIV-associated myelopathy,HIV-associated myopathy, immune reconstitution inflammatory syndrome,inflammatory demyelinating polyneuropathy, mononeuropathy multiplex,primary CNS lymphoma (PCNSL), progressive multifocal leukoencephalopathy(PML), and progressive polyradiculopathy.

HIV-associated dementia (HAD) occurs primarily in subjects with moreadvanced HIV infection (AIDS). Symptoms include, behavioral changes, anda gradual decline in cognitive function, including trouble withconcentration, memory, and attention. Subjects with HAD also showprogressive slowing of motor function and loss of dexterity andcoordination. When left untreated, HAD can be fatal. It is rare whenanti-retroviral therapy is used.

In some embodiments, cognitive deficits that may respond to DON or DONderivatives are not caused by virus infection but may be distallyrelated to (caused by) factors believed to cause neurologic diseases,such as AD, MS, PD and schizophrenia.

In some embodiments, the cognitive deficit is due to, or associatedwith, a neurodegenerative disorder. A “neurodegenerative disorder” is adisease, disorder, or condition that is characterized by the progressiveloss of the structure or function of neurons (e.g., degeneration ordysfunction of neurons or other neural cells). Some aspects of thepresently disclosed subject matter relate to correcting cognitivedefects associated with neurodegenerative diseases, disorders, orconditions including, but not limited to, glaucoma, andneurodegenerative diseases, disorders, or conditions of the nervoussystems, such as or associated with amyotrophic lateral sclerosis (ALS),trigeminal neuralgia, glossopharyngeal neuralgia, Bell's Palsy,myasthenia gravis, muscular dystrophy, progressive muscular atrophy,primary lateral sclerosis (PLS), pseudobulbar palsy, progressive bulbarpalsy, spinal muscular atrophy, inherited muscular atrophy, invertebratedisk syndromes, cervical spondylosis, plexus disorders, thoracic outletdestruction syndromes, peripheral neuropathies, porphyria, Alzheimer'sdisease, Huntington's disease, Parkinson's disease, Parkinson's-plusdiseases, multiple system atrophy, progressive supranuclear palsy,corticobasal degeneration, dementia with Lewy bodies, frontotemporaldementia, demyelinating diseases, Guillain-Barre syndrome, multiplesclerosis, Charcot-Marie-Tooth disease, prion diseases,Creutzfeldt-Jakob disease, Gerstmann-Straussler-Scheinker syndrome(GSS), fatal familial insomnia (FFI), bovine spongiform encephalopathy(BSE), Pick's disease, and epilepsy.

In particular embodiments, the cognitive deficit is due to, orassociated with, multiple sclerosis.

In an aspect, the presently disclosed subject matter provides a methodof treating a cognitive deficit in a subject having multiple sclerosis,the method comprising administering to the subject at least oneglutamine antagonist, or prodrug or analog thereof, in an amounteffective to treat the cognitive deficit.

In another aspect, the presently disclosed subject matter involves theuse of at least one glutamine antagonist, or prodrug or analog thereof,for treating a cognitive deficit in a subject having multiple sclerosis.

In yet other aspects, the presently disclosed subject matter provides apharmaceutical composition comprising at least one glutamine antagonist,or a prodrug or analog thereof, in an amount effective to treat acognitive deficit in a subject having multiple sclerosis, and apharmaceutically acceptable carrier, diluent, or excipient.

In an aspect, the presently disclosed subject matter provides a methodof preventing a cognitive deficit in a subject having multiplesclerosis, the method comprising administering to the subject at leastone glutamine antagonist, or prodrug or analog thereof, in an amounteffective to prevent the cognitive deficit.

In another aspect, the presently disclosed subject matter involves theuse of at least one glutamine antagonist, or prodrug or analog thereof,for preventing a cognitive deficit in a subject having multiplesclerosis.

In yet other aspects, the presently disclosed subject matter provides apharmaceutical composition comprising at least one glutamine antagonist,or a prodrug or analog thereof, in an amount effective to prevent acognitive deficit in a subject having multiple sclerosis, and apharmaceutically acceptable carrier, diluent, or excipient.

In an aspect, the presently disclosed subject matter provides a methodof treating a cognitive deficit in a subject having multiple sclerosis,the method comprising administering to the subject a prodrug of at leastone glutamine antagonist in an amount effective to treat the cognitivedeficit.

In another aspect, the presently disclosed subject matter involves theuse of a prodrug of at least one glutamine antagonist for treating acognitive deficit in a subject having multiple sclerosis.

In yet other aspects, the presently disclosed subject matter provides apharmaceutical composition comprising at least one glutamine antagonist,or a prodrug or analog thereof, in an amount effective to treat acognitive deficit in a subject having multiple sclerosis, and apharmaceutically acceptable carrier, diluent, or excipient.

Some aspects of the presently disclosed subject matter relate tocorrecting cognitive defects associated other neurodegenerativediseases, disorders, or conditions of the nervous systems, such as orassociated with alcoholism, Alexander's disease, Alper's disease, ataxiatelangiectasia, Batten disease (also known asSpielmeyer-Vogt-Sjogren-Batten disease), Canavan disease, Cockaynesyndrome, diabetic neuropathy, frontotemporal lobar degeneration,HIV-associated dementia, Kennedy's disease, Krabbe's disease,neuroborreliosis, Machado-Joseph disease (Spinocerebellar ataxia type3), wet or dry macular degeneration, Niemann Pick disease,Pelizaeus-Merzbacher Disease, photoreceptor degenerative diseases, suchas retinitis pigmentosa and associated diseases, Refsum's disease,Sandhoffs disease, Schilder's disease, subacute combined degeneration ofspinal cord secondary to pernicious anemia,Spielmeyer-Vogt-Sjogren-Batten disease (also known as Batten disease),spinocerebellar ataxia (multiple types with varying characteristics),Steele-Richardson-Olszewski disease, and tabes dorsalis.

Some aspects of the presently disclosed subject matter relate tocorrecting cognitive defects associated with a wide range of geneticbrain diseases. For example, genetic brain diseases may include but arenot limited to Adrenoleukodystrophy, Agenesis of the Corpus Callosum,Aicardi Syndrome, Alpers' Disease. Alzheimer's Disease, Barth Syndrome,Batten Disease, CADASIL, Cerebellar Degeneration, Fabry's Disease,Gerstmann-Straussler-Scheinker Disease, Huntington's Disease and otherTriplet Repeat Disorders, Leigh's Disease, Lesch-Nyhan Syndrome, MenkesDisease, Mitochondrial Myopathies and NINDS Colpocephaly.

Some aspects of the presently disclosed subject matter relate tocorrecting cognitive defects associated with one or more conditions thatare secondary to a disease, disorder, condition, or therapy having aprimary effect outside of the nervous system selected from the groupconsisting of: peripheral neuropathy or neuralgia caused by diabetes,cancer, hepatitis, hepatic encephalopathy, kidney dysfunction, Coloradotick fever, diphtheria, leprosy, Lyme disease, polyarteritis nodosa,rheumatoid arthritis, sarcoidosis, Sjogren syndrome, syphilis, systemiclupus erythematosus, viral encephalitis, and amyloidosis. In someembodiments, the cognitive deficit is associated with hepaticencephalopathy. In some embodiments, the cognitive deficit is associatedwith viral encephalitis.

Some aspects of the presently disclosed subject matter relate tocorrecting cognitive defects associated with a neurodegenerativedisease, disorder, or condition associated with pain selected from thegroup consisting of chronic pain, fibromyalgia, spinal pain, carpeltunnel syndrome, pain from cancer, arthritis, sciatica, headaches, painfrom surgery, muscle spasms, back pain, visceral pain, pain from injury,dental pain, neuralgia, such as neurogenic or neuropathic pain, nerveinflammation or damage, shingles, herniated disc, a torn ligament, anddiabetes.

Some aspects of the presently disclosed subject matter relate tocorrecting cognitive defects associated with a neurodegenerativedisease, disorder, or condition that is associated with one or moreinjuries to the nervous system. In particular embodiments, the one ormore injuries to the nervous system is related to nerve damage caused byexposure to one or more agents selected from the group consisting oftoxic compounds, heavy metals, industrial solvents, drugs,chemotherapeutic agents, dapsone, cholesterol lowering drugs, heart orblood pressure medications, and metronidazole.

In more particular embodiments, the one or more injuries to the nervoussystem is related to nerve damage caused by one or more conditionsselected from the group consisting of burn, wound, surgery, accidents,ischemia, prolonged exposure to cold temperature, stroke, intracranialhemorrhage, and cerebral hemorrhage.

Some aspects of the presently disclosed subject matter relate tocorrecting cognitive defects associated with a psychiatric disorder. Inparticular embodiments, the psychiatric disorder is selected from thegroup consisting of schizophrenia, delusional disorder, schizoaffectivedisorder, schizophreniform, shared psychotic disorder, psychosis,paranoid personality disorder, schizoid personality disorder, borderlinepersonality disorder, anti-social personality disorder, narcissisticpersonality disorder, obsessive-compulsive disorder, delirium, dementia,mood disorders, bipolar disorder, depression, stress disorder, panicdisorder, agoraphobia, social phobia, post-traumatic stress disorder,anxiety disorder, and impulse control disorders.

In another embodiment, the disclosure provides a method for treating asubject having a cognitive deficit or psychiatric disorder, the methodcomprising administering to the subject a compound, or apharmaceutically acceptable salt thereof, having formula (I):

wherein:

-   -   X is selected from the group consisting of a bond, —O—, and        —(CH₂)_(n)—, wherein n is an integer selected from the group        consisting of 1, 2, 3, 4, 5, 6, 7, and 8;    -   R₁ is selected from the group consisting of C₁₋₆ alkyl and        substituted C₁₋₆ alkyl;    -   R₂ is —C(═O)—O—(CR₃R₄)_(m)—O—C(═O)—R₁₀;    -   R₂′ is selected from the group consisting of H, C₁-C₆ alkyl, and        substituted C₁-C₆ alkyl;    -   each R₃ and R₄ is independently H, C₁-C₆ alkyl, substituted        C₁-C₆ alkyl, aryl, substituted aryl, —(CR₃R₄)_(m)—NR₅R₆, or

-   -   m is an integer selected from the group consisting of 1, 2, 3,        4, 5, 6, 7, and 8;    -   R₅ and R₆ is independently H or alkyl; and    -   R₁₀ is selected from the group consisting of alkyl, substituted        alkyl, cycloalkyl, substituted cycloalkyl, monosaccharide,        acylated monosaccharide, aryl, substituted aryl, heteroaryl, and        substituted heteroaryl,    -   in an amount effective to treat the cognitive deficit or        psychiatric disorder.

In another embodiment, the disclosure provides a method for treating asubject having a cognitive deficit or psychiatric disorder, the methodcomprising administering to the subject a compound, or apharmaceutically acceptable salt thereof, having formula (I), wherein Xis —CH₂—. In another embodiment, X is —O—. In another embodiment, R₁ isselected from the group consisting of methyl, ethyl, isopropyl,cyclopentyl, cyclohexyl, trimethylammonium, triethylammonium,tri(hydroxyethyl)ammonium, tripropylammonium, andtri(hydroxypropyl)ammonium. In another embodiment, m is 1; each R₃ andR₄ are independently H, C₁-C₆ alkyl, aryl or substituted aryl; and R₁₀is selected from the group consisting of alkyl, substituted alkyl,cycloalkyl, substituted cycloalkyl, aryl, substituted aryl, heteroaryl,and substituted heteroaryl.

In another embodiment, the disclosure provides a method for treating asubject having a cognitive deficit or psychiatric disorder, the methodcomprising administering to the subject a compound, or apharmaceutically acceptable salt thereof, having formula (II):

wherein:

-   -   R₁ is selected from the group consisting of H and C₁₋₆ alkyl;    -   R₃ and R₄ are independently selected from the group consisting        of H, C₁-C₆ alkyl, substituted C₁-C₆ alkyl, aryl, and        substituted aryl; and    -   R₁₀ is C₁₋₆ alkyl.

In another embodiment, the disclosure provides a method for treating asubject having a cognitive deficit or psychiatric disorder, the methodcomprising administering to the subject a compound, or apharmaceutically acceptable salt thereof, having formula (II), whereinR₁ is selected from the group consisting of methyl, ethyl, andisopropyl. In another embodiment, R₃ is H and R₄ is selected from thegroup consisting of methyl and phenyl. In another embodiment, R₃ isselected from the group consisting of methyl and phenyl, and R₄ is H. Inanother embodiment, R₁₀ is selected from the group consisting ofisopropyl and tert-butyl.

In another embodiment, the disclosure provides a method for treating asubject having a cognitive deficit or psychiatric disorder, the methodcomprising administering to the subject a compound, or apharmaceutically acceptable salt thereof, selected from the groupconsisting of:

In another embodiment, the disclosure provides a method for treating asubject having a cognitive deficit or psychiatric disorder, the methodcomprising administering to the subject a compound, or apharmaceutically acceptable salt thereof, selected from the groupconsisting of:

In another embodiment, the disclosure provides a method for treating asubject having a cognitive deficit, the method comprising administeringto the subject a compound having formula (I) or formula (II), or apharmaceutically acceptable salt thereof.

In another embodiment, the disclosure provides a method for treating asubject having a cognitive deficit, the method comprising administeringto the subject a compound having formula (I) or formula (II), or apharmaceutically acceptable salt thereof, wherein the cognitive deficitis characterized by impairment of the mental processes of perception,learning, memory, judgment, and/or reasoning.

In another embodiment, the disclosure provides a method for treating asubject having a cognitive deficit, the method comprising administeringto the subject a compound having formula (I) or formula (II), or apharmaceutically acceptable salt thereof, wherein the cognitive deficitis due to a viral infection. In another embodiment, the cognitivedeficit is due to the human immunodeficiency virus (HIV). In anotherembodiment, the virus is latent.

In another embodiment, the disclosure provides a method for treating asubject having a cognitive deficit, the method comprising administeringto the subject a compound having formula (I) or formula (II), or apharmaceutically acceptable salt thereof, wherein the cognitive deficitis selected from the group consisting of asymptomatic neurocognitiveimpairment (ANI) and mild neurocognitive disorder (MND).

In another embodiment, the disclosure provides a method for treating asubject having a cognitive deficit, the method comprising administeringto the subject a compound having formula (I) or formula (II), or apharmaceutically acceptable salt thereof, wherein the cognitive deficitis associated with a neurodegenerative disorder.

In another embodiment, the disclosure provides a method for treating asubject having a cognitive deficit, the method comprising administeringto the subject a compound having formula (I) or formula (II), or apharmaceutically acceptable salt thereof, wherein the cognitive deficitis associated with a psychiatric disorder.

In another embodiment, the disclosure provides a method for treating asubject having a cognitive deficit, the method comprising administeringto the subject a compound having formula (I) or formula (II), or apharmaceutically acceptable salt thereof, wherein the cognitive deficitis associated with multiple sclerosis.

In another embodiment, the disclosure provides a method of treating asubject having a psychiatric disorder, the method comprisingadministering to the subject a compound having formula (I) or formula(II), or a pharmaceutically acceptable salt thereof. In anotherembodiment, the psychiatric disorder is a mood disorder.

In another embodiment, the disclosure provides a method for treating asubject having a cognitive deficit, the method comprising administeringto the subject a compound having formula (I) or formula (II), or apharmaceutically acceptable salt thereof, wherein the cerebrospinalfluid (CSF)-to-plasma concentration ratio of 6-diazo-5-oxo-L-norleucine(DON) in the subject following administration is about 0.20 to about5.0. In another embodiment, CSF-to-plasma concentration ratio of DON inthe subject following administration is about 0.25 to about 5.0. Inanother embodiment, CSF-to-plasma concentration of DON in the subjectfollowing administration is about 0.30 to about 5.0. In anotherembodiment, CSF-to-plasma concentration ratio of DON in the subjectfollowing administration is about 0.35 to about 5.0. In anotherembodiment, CSF-to-plasma concentration ratio of DON in the subjectfollowing administration is about 0.35 to about 2.0. In anotherembodiment, CSF-to-plasma concentration ratio of DON in the subjectfollowing administration is about 0.35 to about 1.0. In anotherembodiment, CSF-to-plasma concentration ratio of DON in the subjectfollowing administration is about 0.35 to about 0.75. In anotherembodiment, CSF-to-plasma concentration ratio of DON in the subjectfollowing administration is about 0.35 to about 0.50.

The disclosure also provides the following particular embodiments.

Embodiment I. A method for treating a subject having a cognitivedeficit, the method comprising administering to the subject at least oneglutamine antagonist in an amount effective to treat the cognitivedeficit.

Embodiment II. The method of Embodiment 1, wherein the cognitive deficitis characterized by impairment of the mental processes of perception,learning, memory, judgment, and/or reasoning.

Embodiment III. The method of Embodiment 1, wherein the cognitivedeficit is due to a viral infection.

Embodiment IV. The method of Embodiment 3, wherein the cognitive deficitis due to the human immunodeficiency virus (HIV).

Embodiment V. The method of Embodiment 4, wherein the virus is latent.

Embodiment VI. The method of Embodiment 1, wherein the cognitive deficitis selected from the group consisting of asymptomatic neurocognitiveimpairment (ANI), mild neurocognitive disorder (MND), and HIV-associateddementia (HAD).

Embodiment VII. The method of Embodiment 1, wherein the cognitivedeficit is associated with a neurodegenerative disorder and/orpsychiatric disorder.

Embodiment VIII. The method of Embodiment 1, wherein the cognitivedeficit is associated with multiple sclerosis.

Embodiment IX. The method of Embodiment 1, wherein the at least oneglutamine antagonist is a glutamine analog.

Embodiment X. The method of Embodiment 1, wherein the at least oneglutamine antagonist is:

-   -   (i) a glutamine analog;    -   (ii) a glutamine analog selected from the group consisting of        acivicin (L-(alpha S,        5S)-alpha-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid),        azaserine, 6-diazo-5-oxo-norleucine (DON), and        5-diazo-4-oxo-L-norvaline (L-DONV);    -   (iii) a prodrug of a glutamine analog; or    -   (iv) a prodrug of acivicin, azaserine, DON, and L-DONV.

Embodiment XI. A pharmaceutical composition comprising at least oneglutamine antagonist, or a prodrug or analog thereof, in an amounteffective to treat a cognitive deficit, and a pharmaceuticallyacceptable carrier, diluent, or excipient.

Embodiment XII. The method of Embodiment 11, wherein the cognitivedeficit is characterized by impairment of the mental processes ofperception, learning, memory, judgment, and/or reasoning.

Embodiment XIII. The method of Embodiment 11, wherein the cognitivedeficit is due to a viral infection.

Embodiment XIV. The method of Embodiment 13, wherein the cognitivedeficit is due to the human immunodeficiency virus (HIV).

Embodiment XV. The method of Embodiment 14, wherein the virus is latent.

Embodiment XVI. The method of Embodiment 11, wherein the cognitivedeficit is selected from the group consisting of asymptomaticneurocognitive impairment (ANI), mild neurocognitive disorder (MND), andHIV-associated dementia (HAD).

Embodiment XVII. The method of Embodiment 11, wherein the cognitivedeficit is associated with a neurodegenerative disorder and/orpsychiatric disorder.

Embodiment XVIII. The method of Embodiment 11, wherein the cognitivedeficit is associated with multiple sclerosis.

Embodiment XIX. The method of Embodiment 11, wherein the at least oneglutamine antagonist is a glutamine analog.

Embodiment XX. The method of Embodiment 11, wherein the at least oneglutamine antagonist is:

-   -   (i) a glutamine analog;    -   (ii) a glutamine analog selected from the group consisting of        acivicin (L-(alpha S,        5S)-alpha-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid),        azaserine, 6-diazo-5-oxo-norleucine (DON), and        5-diazo-4-oxo-L-norvaline (L-DONV);    -   (iii) a prodrug of a glutamine analog; or    -   (iv) a prodrug of acivicin, azaserine, DON, and L-DONV

As used herein, the term “glutamine antagonist” refers to an agent thatblocks or interferes with the synthesis or use of glutamine in a cell,and preferably in a cell that is part of a living organism. When it issaid that the glutamine antagonist interferes with the synthesis ofglutamine, it is meant that the antagonist acts to reduce the amount orrate of glutamine synthesis to less than the amount or rate that wouldbe experienced in the absence of the glutamine antagonist. When it issaid that the glutamine antagonist interferes with the use of glutamine,it is meant that the antagonist acts to inhibit or block a metabolicpathway downstream of glutamine, that is, a pathway in which glutamineacts as a precursor of one or more non-glutamine compounds, or that theantagonist acts to deplete glutamine in a cell or an organism byreacting the glutamine to form a non-glutamine product, or by reversiblyor irreversibly binding with glutamine to reduce its availability.

In some embodiments, a glutamine antagonist is a compound that inhibitsthe synthesis of glutamine. Examples of compounds having this activityinclude inhibitors of glutamine synthase (EC 6.3.1.2), such asL-methionine-DL-sulfoximine, and phosphinothricin; inhibitors ofglutamate synthase (EC 1.4.1.13); inhibitors ofamidophosphoribosyltransferase (EC 2.4.2.14); and inhibitors ofglutamate dehydrogenase; and mixtures of any two or more of these.

In some embodiments, a glutamine antagonist is a glutamine depletingenzyme. Examples of such enzymes include carbamoyl-phosphate synthase(EC 6.3.5.5), glutamine-pyruvate transaminase (EC 2.6.1.15),glutamine-tRNA ligase (EC 6.1.1.18), glutaminase (EC 3.5.1.2),D-glutaminase (EC 3.5.1.35), glutamine N-acyltransferase (EC2.3.1.68),glutaminase-asparaginase (in particular glutaminase-asparaginase ofPseudomonas 7a and Acinatobacter sp.), and mixtures of any two or moreof these.

In some embodiments, a glutamine antagonist is a compound that reactswith glutamine under intracellular conditions to form a non-glutamineproduct. An example of a compound having this property is phenylbutyrate(see Darmaun et al., Phenylbutyrate-induce glutamine depletion inhumans: effect on leucine metabolism, pp. E801-E807, in GlutamineDepletion and Protein Catabolism, Am. Physiol. Soc. (1998)). Anotherexample of a glutamine antagonist having this characteristic isphenylacetate (see, U.S. Pat. No. 6,362,226), which is incorporatedherein by reference in its entirety.

In some embodiments, a glutamine antagonist is a compound that inhibitsglutamine uptake by cells. Examples of compounds having this propertyinclude alpha-methylaminoisobutyric acid (inhibits GynT plasma membraneglutamine transporter; see, Varoqui et al., J. Biol. Chem.,275(6):4049-4054 (2000), wortmannin, and LY-294002 (inhibits hepaticglutamine transporter; see, Pawlik et al., Am. J. Physiol. Gastrointest.Liver Physiol., 278:G532-G541 (2000)).

In some embodiments, a glutamine antagonist is a glutamine bindingcompound that reduces the biological availability of glutamine.

In some embodiments, a glutamine antagonist is a glutamine analog thatinterferes with a glutamine metabolic pathway. Examples of compoundsthat can act in this manner include acivicin (L-(alphaS,5S)-alpha-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid), DON(6-diazo-5-oxo-L-norleucine), azaserine, azotomycin, chloroketone(L-2-amino-4-oxo-5-chloropentanoic acid),N³-(4-methoxyfumaroyl)-L-2,3-diaminopropanoic acid (FMDP) (inactivatesglucosamine-6-phosphate synthase (EC 2.6.1.16), see, Zgbdka et al.,Microbiology, 147:1955-1959 (2001)), (3S,4R)-3,4-dimethyl-L-glutamine,(3S,4R)-3,4-dimethyl-L-pyroglutamic acid (see, Acevedo et al.,Tetrahedron., 57:6353-6359 (2001)),1,5-N,N′-disubstituted-2-(substituted benzenesulphonyl) glutamamides(see, Srikanth et al., Bioorganic and Medicinal Chemistry, (2002)), or amixture of any two or more of these. In some embodiments, at least oneglutamine antagonist is a glutamine analog. In some embodiments, atleast one glutamine antagonist is selected from the group consisting ofacivicin (L-(alpha S,5S)-alpha-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid), azaserine,and 6-diazo-5-oxo-norleucine (DON).

In some embodiments, at least one glutamine antagonist is a prodrug of aglutamine analog. In some embodiments, at least one glutamine antagonistis a prodrug of acivicin (L-(alpha S,5S)-alpha-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid), azaserine,and 6-diazo-5-oxo-norleucine (DON).

In some aspects, a prodrug of a glutamine antagonist, or apharmaceutically acceptable salt or ester thereof has a structure offormula (I):

wherein: X is selected from the group consisting of a bond, —O—, and—(CH₂)_(n)—, wherein n is an integer selected from the group consistingof 1, 2, 3, 4, 5, 6, 7, and 8; R₁ is selected from the group consistingof H and a first prodrug-forming moiety capable of forming a salt or anester; and R₂ is H or a second prodrug-forming moiety capable of formingan amide linkage, a carbamate linkage, a phosphoramidate linkage or aphosphorodiamidate linkage with the nitrogen adjacent to R₂; R₂′ isselected from the group consisting of H, C₁-C₆ alkyl, substituted C₁-C₆alkyl, or R₂ and R₂′ together form a ring structure comprising—C(═O)-G-C(═O)—, wherein G is selected from the group consisting ofC₁-C₈ alkylene, C₁-C₈ heteroalkylene, C₅-C₈ cycloalkylene, C₆-C₁₂arylene, C₅-C₁₄ heteroarylene, bivalent C₄-C₁₀ heterocycle, each ofwhich can be optionally substituted; or R₁ and R₂′ together form a 4- to6-membered heterocylic ring comprising the oxygen atom adjacent to R₁and the nitrogen atom adjacent to R₂′; provided that the compound has atleast one prodrug-forming moiety selected from the group consisting ofthe first and the second prodrug-forming moieties.

As used herein, the term “amide linkage” comprises a structurerepresented by the formula:

wherein R_(v) is selected from the group consisting of alkyl,substituted alkyl, cycloalkyl, substituted cycloalkyl, aryl, substitutedaryl, aralkyl, substituted aralkyl, heterocyclyl, substitutedheterocyclyl, alkenyl, substituted alkenyl, cycloalkenyl, substitutedcycloalkenyl, alkylamine, substituted alkylamine, heteroaryl, andsubstituted heteroaryl.

As used herein, the term “carbamate linkage” comprises a structurerepresented by the formula:

wherein R_(w) is selected from the group consisting of alkyl,substituted alkyl, cycloalkyl, substituted cycloalkyl, aryl, substitutedaryl, aralkyl, substituted aralkyl, heterocyclyl, substitutedheterocyclyl, alkenyl, substituted alkenyl, cycloalkenyl, substitutedcycloalkenyl, alkylamine, substituted alkylamine, heteroaryl, andsubstituted heteroaryl.

As used herein, the term “phosphoramidate linkage” comprises a structurerepresented by the formula:

wherein R_(x) and R_(x)′ are each independently selected from the groupconsisting of alkyl, substituted alkyl, cycloalkyl, substitutedcycloalkyl, aryl, substituted aryl, aralkyl, substituted aralkyl,heterocyclyl, substituted heterocyclyl, alkenyl, substituted alkenyl,cycloalkenyl, substituted cycloalkenyl, alkylamine, substitutedalkylamine, heteroaryl, and substituted heteroaryl.

As used herein, the term “phosphorodiamidate linkage” comprises astructure represented by the formula:

wherein R_(y) and R_(z) are each independently selected from the groupconsisting of H, alkyl, substituted alkyl, cycloalkyl, substitutedcycloalkyl, heterocyclyl, substituted heterocyclyl, alkenyl, substitutedalkenyl, cycloalkenyl, substituted cycloalkenyl, —(CR₃R₄)_(m)—Z,—(CR₃R₄)_(m)—Q—Z, aryl, substituted aryl, alkylamine, substitutedalkylamine, heteroaryl, substituted heteroaryl, and

In some embodiments, X is —CH₂—, and n is 1.

In other embodiments, X is —O—. In some embodiments, the prodrugcompound has both the first prodrug-forming moiety and the secondprodrug-forming moiety. In some embodiments, the glutamine analog is aglutamine antagonist, i.e., the prodrug is a prodrug of a glutamineanalog that antagonizes a glutamine pathway. Exemplary glutamineantagonists include, without limitation, 6-diazo-5-oxo-norleucine (DON),and aza-serine, and 5-diazo-4-oxo-L-norvaline (L-DONV).

In some embodiments, the presently disclosed subject matter provides aprodrug of DON. In some embodiments, the prodrug of DON has a structureof formula (I). In some embodiments, the presently disclosed subjectmatter provides a prodrug of L-DONV. In some embodiments, the prodrug ofL-DONV has a structure of formula (I). In some embodiments, thepresently disclosed subject matter provides a prodrug of azaserine. Insome embodiments, the prodrug of azaserine has a structure of formula(I).

In some embodiments, R₁ of formula (I) comprises a residue PRO₁ of theprodrug-forming moiety, which, together with a basic moiety and theterminal hydroxyl group forms a salt.

In some embodiments, R₁ of formula (I) comprises a residue PRO₁ of theprodrug-forming moiety, which, together with an alkyl group and theoxygen of an adjoining hydroxyl group forms an ester.

In some embodiments, R₁ of formula (I) comprises a residue PRO₁ of theprodrug-forming moiety, which, together with an alkyl group and thenitrogen adjoining the R₂′ group, forms an azlactone or an oxazolidone.

In some embodiments, R₁ of formula (I) is selected from the groupconsisting of H, alkyl, substituted alkyl, cycloalkyl, substitutedcycloalkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl,cycloalkenyl, substituted cycloalkenyl, tri(hydrocarbyl)ammonium, andtetra(hydrocarbyl)ammonium. Preferred alkyl group, cycloalkyl group,alkenyl group, alkynyl group, and cycloalkenyl group substituentsinclude alkyl, substituted alkyl, halo, arylamino, acyl, hydroxyl,aryloxyl, alkoxyl, alkylthio, arylthio, aralkyloxyl, aralkylthio,carboxyl, alkoxycarbonyl, oxo, and cycloalkyl.

In some embodiments, R₁ of formula (I) is not H. In some embodiments, R₁of formula (I) is not H when R₂ and R₂ are H. In some embodiments, R₂and R₂ of formula (I) are each H when and R₁ is not H.

In some embodiments, R₁ of formula (I) is selected from the groupconsisting of a C₁₋₆ straight-chain alkyl, a substituted C₁₋₆straight-chain alkyl, a C₁₋₆ branched alkyl, a substituted C₁₋₆ branchedalkyl, tri(C₁-C₈-alkyl)ammonium, tetra(C₁-C₈-alkyl)ammonium,triphenylammonium, tri(hydroxy-C₁-C₈-alkyl)ammonium, andtetra(hydroxy-C₁-C₈-alkyl)ammonium.

In some embodiments, R₁ of formula (I) is selected from the groupconsisting of methyl, ethyl, isopropyl, cyclopentyl, cyclohexyl,trimethylammonium, triethylammonium, tri(hydroxyethyl)ammonium,tripropylammonium, and tri(hydroxypropyl)ammonium. In some embodiments,R₁ of formula (I) is methyl. In some embodiments, R₁ of formula (I) isethyl. In some embodiments, R₁ of formula (I) is isopropyl.

In some embodiments, R₂ of formula (I) comprises a residue PRO₂ of thesecond prodrug-forming moiety, which, together with a carbonyl, oxycarbonyl, or phosphonyl group and the nitrogen of the adjoining NH,forms an amide, a carbamate, phosphoramidate, or phosphorodiamidatelinkage.

In some embodiments, R₂ of formula (I) comprises a moiety selected fromthe group consisting of an amino acid, an N-substituted amino acid, apeptide, a substituted peptide, a monocyclic ring, a substitutedmonocyclic ring, a bicyclic ring, a substituted bicyclic ring, a purinenucleoside, a substituted purine nucleoside, a pyrimidine nucleoside,and a substituted pyrimidine nucleoside.

In some embodiments, R₂ of formula (I) is selected from the groupconsisting of H, alkyl, —C(═O)—Ar, —C(═O)—Y—(CR₃R₄)_(m)—Ar,—C(═O)—Y—(CR₃R₄)_(m)—NR₅R₆, —P(═O)(OR₇)_(n)(NHR₉)_(o),—C(═O)—Y—(CR₃R₄)_(m)—Ar—O—C(═O)—R₈, —C(═O)—Y—(CR₃R₄)_(m)—Ar—O—R₈,—C(═O)—O—(CR₃R₄)_(m)—O—C(═O)—R₁₀, —C(═O)—O—R₉,—C(═O)—Y—(CR₃R₄)_(m)—Ar—O—C(═O)—Ar, and —C(═O)—Y—(CR₃R₄)_(m)—Ar—NR₅R₆;wherein: Y is —O— or a bond; m is an integer selected from the groupconsisting of 1, 2, 3, 4, 5, 6, 7, and 8; each n and o is an integerfrom 0 to 2 provided that the sum of n and o is 2; R₃ and R₄ isindependently H, C₁-C₆ alkyl or substituted C₁-C₆ alkyl, aryl orsubstituted aryl, —(CR₃R₄)_(m)—NR₅R₆, or

each R₅ and R₆ is independently H, alkyl, —C(═O)—(CR₃R₄)_(m),—C(═O)—(NR₅R₆)H, or —C(═O)—(CR₃R₄)_(m)—NR₅R₆; each R₇ is independentlyselected from the group consisting of H, alkyl, substituted alkyl,cycloalkyl, substituted cycloalkyl, heterocyclyl, substitutedheterocyclyl, alkenyl, substituted alkenyl, cycloalkenyl, substitutedcycloalkenyl, —(CR₃R₄)_(m)—Z, —(CR₃R₄)_(m)-Q-Z, wherein Q is amonosaccharide, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, and wherein Z is;

or wherein R₇ together with the oxygen atom to which it is attachedforms a purine or pyrimidine nucleoside; each R₉ is independentlyselected from the group consisting of H, alkyl, substituted alkyl,cycloalkyl, substituted cycloalkyl, heterocyclyl, substitutedheterocyclyl, alkenyl, substituted alkenyl, cycloalkenyl, substitutedcycloalkenyl, —(CR₃R₄)_(m)—Z, aryl, substituted aryl, heteroaryl,substituted heteroaryl, and

wherein R₁ and X are as defined above, provided that R₁ is not H;

each R₈ is independently alkyl, substituted alkyl, cycloalkyl,substituted cycloalkyl, monosaccharide, acylated monosaccharide, aryl,substituted aryl, heteroaryl, substituted heteroaryl; each R₁₀ isindependently alkyl, substituted alkyl, cycloalkyl, substitutedcycloalkyl, monosaccharide, acylated monosaccharide, aryl, substitutedaryl, heteroaryl, substituted heteroaryl; and Ar is aryl, substitutedaryl, heteroaryl, or substituted heteroaryl. It should be appreciatedthat in addition to substitutions on the amino group of Z, one or moresubstitutions R₃, R₄, R₅, and/or R₆ can be made to the 5 or 6 memberedrings of Z.

Structures of representative DON prodrugs are provided in Table 1.

TABLE 1 Structures of Representative DON Prodrugs IOCB No./ Compound No.Structure MW Compound 1 (DON)

171.15 Compound 3

213.24 Compound 4

445.41 Compound 6

391.38 Compound 7

564.53 Compound 9

326.39 Compound 11

439.55 Compound 13

369.18 Compound 14a ^(#)

385.41 Compound 14b ^(#)

Compound 15

371.39 Compound 17

375.33 Compound 20

199.21 Compound 22

270.28 Compound 23

343.42 Compound 25

312.36 Compound 26

385.50 Compound 28

425.52 Compound 29

329.31 Compound 30

343.33 Compound 31

357.37 Compound 32

371.39 Compound 34

385.42 Compound 35

327.25 Compound 36

355.30 Compound 38a

399.45 Compound 38

399.45 Compound 40

413.47 Compound 42

371.39 Compound 44

2.44 Compound 47

447.49 Compound 49

357.36 Compound 51

618.69 Compound 52

660.73 Compound 56

469.54 Compound 57

511.58 Compound 59

511.48 Compound 60a

464.19 Compound 60

464.19 A

618.54 B

602.54 C

530.47 D

334.38 E

484.51 F

525.51 G

509.51 LTP 073

255.23 JAM0351

693.66 JAM0359

679.63 ^(#) A diastereomeric mixture of isopropyl(2S)-6-diazo-5-oxo-2-(((1-(pivaloyloxy)ethoxy)carbonyl)amino)hexanoatewas prepared and separated by column chromatography to give isopropyl(S)-6-diazo-5-oxo-2-((((S)-1-(pivaloyloxy)ethoxy)carbonyl)amino)hexanoateand isopropyl(S)-6-diazo-5-oxo-2-((((R)-1-(pivaloyloxy)ethoxy)carbonyl)amino)hexanoate.The S,S-isomer was arbitrarily designated compound 14a, and theS,R-isomer was arbitrarily designated compound 14b. The actualstereochemistry of the acetal methyl group was not determined. Thediastereoisomer that was arbitrarily designated compound 14b was used inthe biological studies described herein. See PCT/US2016/044767 (WO2017/023774 A1), which is fully incorporated by reference herein.

As described herein, compound 14b demonstrated unexpected stability inhuman and monkey plasma and achieved an unexpected enhanced CSF/plasmaratio versus DON in monkeys (10 fold). In addition, compound 47 achievedan unexpected enhanced CSF/plasma ratio versus DON in swine (15 fold).Thus, these compounds are uniquely useful for the treatment of cognitivedeficits that require enhanced levels of DON in the brain.

In general, the presently disclosed methods result in a decrease in theseverity of a condition, disease, or disorder (e.g., a cognitivedeficit) in a subject. The term “decrease” is meant to inhibit,suppress, attenuate, diminish, arrest, or stabilize a symptom of thecondition, disease, or disorder. As used herein, the terms “treat,”“treating,” “treatment,” and the like refer to reducing or amelioratinga disease or condition, and/or symptoms associated therewith. It will beappreciated that, although not precluded, treating a disease orcondition does not require that the disorder, condition or symptomsassociated therewith be completely eliminated.

The terms “subject” and “patient” are used interchangeably herein. Thesubject treated by the presently disclosed methods, uses, glutamineantagonists and compositions comprising those glutamine antagonists intheir many embodiments is desirably a human subject, although it is tobe understood that the methods described herein are effective withrespect to all vertebrate species, which are intended to be included inthe term “subject.” Accordingly, a “subject” can include a human subjectfor medical purposes, such as for the treatment of an existing conditionor disease or the prophylactic treatment for preventing the onset of acondition or disease, or an animal subject for medical, veterinarypurposes, or developmental purposes. Suitable animal subjects includemammals including, but not limited to, primates, e.g., humans, monkeys,apes, and the like; bovines, e.g., cattle, oxen, and the like; ovines,e.g., sheep and the like; caprines, e.g., goats and the like; porcines,e.g., pigs, hogs, and the like; equines, e.g., horses, donkeys, zebras,and the like; felines, including wild and domestic cats; canines,including dogs; lagomorphs, including rabbits, hares, and the like; androdents, including mice, rats, and the like. An animal may be atransgenic animal. In some embodiments, the subject is a humanincluding, but not limited to, fetal, neonatal, infant, juvenile, andadult subjects. Further, a “subject” can include a patient afflictedwith or suspected of being afflicted with a condition or disease.

The terms “decrease,” “reduced,” “reduction,” “decrease,” or “inhibit”are all used herein generally to mean a decrease by a statisticallysignificant amount. However, for avoidance of doubt, “reduced,”“reduction,” “decrease,” or “inhibit” means a decrease by at least 10%as compared to a reference level, for example a decrease by at leastabout 20%, or at least about 30%, or at least about 40%, or at leastabout 50%, or at least about 60%, or at least about 70%, or at leastabout 80%, or at least about 90%, where the decrease is less than 100%.In one embodiment, the decrease includes a 100% decrease (e.g. absentlevel as compared to a reference sample), or any decrease between10-100% as compared to a reference level.

The terms “increased,” “increase,” “enhance,” or “activate” are all usedherein to generally mean an increase by a statically significant amount;for the avoidance of any doubt, the terms “increased,” “increase,”“enhance,” or “activate” means an increase of at least 10% as comparedto a reference level, for example an increase of at least about 20%, orat least about 30%, or at least about 40%, or at least about 50%, or atleast about 60%, or at least about 70%, or at least about 80%, or atleast about 90% or up to and including a 100% increase or any increasebetween 10-100% as compared to a reference level, or at least about a2-fold, or at least about a 3-fold, or at least about a 4-fold, or atleast about a 5-fold or at least about a 10-fold increase, or anyincrease between 2-fold and 10-fold or greater as compared to areference level.

The term “statistically significant” or “significantly” refers tostatistical significance and generally means a two standard deviation(2SD) below normal, or lower, concentration of the marker. The termrefers to statistical evidence that there is a difference. It is definedas the probability of making a decision to reject the null hypothesiswhen the null hypothesis is actually true. The decision is often madeusing the p-value.

Generally, at least one glutamine antagonist described herein can beused in combination with an additional therapeutic agent (e.g., apharmaceutically active agent, e.g., a drug approved by a regulatoryagency). The therapeutic agent may act synergistically with theglutamine antagonist described herein, or they may independently exerttheir intended effects. The disclosure contemplates any therapeuticagent which a skilled artisan would use in connection with a method,use, or composition described herein. Examples of therapeutic agentscontemplated for use in the presently disclosed methods, uses andcompositions in combination with the glutamine antagonists include, butare not limited to, antiviral agents, immunotherapeutic agents,anti-inflammatory agents, neuroprotective agents, neuroregenerativeagents, neurotrophic factors, stem and progenitor cells used to replaceand/or repair endogenous populations of abnormal, harmful, or unhealthycells, and vaccines.

Exemplary classes of antiviral agents of use herein include, withoutlimitation, antiviral boosters, antiviral combinations, antiviralinterferons, chemokine receptor antagonists, integrase strand transferinhibitors, NNRTIs, NS5A inhibitors, nucleoside reverse transcriptaseinhibitors (NRTIs), protease inhibitors, and purine nucleosides.

Examples of antiviral boosters of use herein include, withoutlimitation, ritonavir, cobicistat, and combinations thereof.

Examples of antiviral combinations of use herein include, withoutlimitation, abacavir and lamivudine (EPZICOM),cobicistat/elvitegravir/emtricitabine/tenofovir (STRIBILD),emtricitabine/tenofovir (TRUVADA), efavirenz/emtricitabine/tenofovir(ATRIPLA), ledipasvir/sofosbuvir (HARVONI),abacavir/lamivudine/zidovudine (TRIZIVIR),emtricitabine/rilpivirine/tenofovir (COMPLERA),abacavir/dolutegravir/lamivudine (TRIUMEQ),dasabuvir/ombitasvir/paritaprevir/ritonavir (VIEKIRA PAK),elbasvir/grazoprevir (ZEPATIER), lamivudine/zidovudine (COMBIVIR),cobicistat/elvitegravir/emtricitabine/tenofovir alafenamide (GENVOYA),cobicistat/darunavir (PREZCOBIX), emtricitabine/tenofovir,emtricitabine/lopinavir/ritonavir/tenofovir,emtricitabine/nelfinavir/tenofovir, lamivudine/raltegravir (DUTREBIS),atazanavir/cobicistat (EVOTAZ), interferon alfa-2b/ribavirin (REBETRON),ombitasvir/paritaprevir/ritonavir (TECHNIVIE), and combinations thereof.

Examples of antiviral interferons of use herein include, withoutlimitation, peginterferon alfa-2a (PEGASYS), peginterferon alfa-2b(PEGINTRON), peginterferon alfa-2b (SYLATRON), and combinations thereof.

An exemplary chemokine receptor antagonist of use herein is maraviroc(SELZENTRY).

Exemplary integrase strand transfer inhibitors of use herein include,without limitation, raltegravir, dolutegravir, elvitegravir, andcombinations thereof.

Exemplary non-nucleoside reverse transcriptase inhibitors (NNRTIs) ofuse herein include, without limitation, nevirapine, etravirine,efavirenz, rilpivirine, delavirdine, nevirapine and combinationsthereof.

An exemplary non-structural protein 5A (NS5A) inhibitor of use herein isdaclatasvir (DAKLINZA).

Exemplary nucleoside reverse transcriptase inhibitors (NRTIs) of useherein include, without limitation, entecavir, lamivudine, adefovir,didanosine, tenofovir, abacavir, lamivudine, zidovudine, stavudine,emtricitabine, zalcitabine, telbivudine, didanosine, and combinationsthereof.

Exemplary protease inhibitors of use herein include, without limitation,boceprevir, simeprevir, telaprevir, lopinavir/ritonavir (KALETRA),fosamprenavir, darunavir, ritonavir, tipranavir, atazanavir, nelfinavir,amprenavir, indinavir, saquinavir, and combinations thereof.

Exemplary purine nucleoside of use herein include, without limitation,ribavirin, valacyclovir, famciclovir, acyclovir, ganciclovir,valganciclovir, cidofovir and combinations thereof.

Other exemplary antiviral agents of use herein include, withoutlimitation, sofosbuvir, enfuvirtide, enfuvirtide, fomivirsen, andcombinations thereof.

As used herein, the term “immunotherapeutic agent” refers to a moleculethat can aid in the treatment of a disease by inducing, enhancing, orsuppressing an immune response in a cell, tissue, organ or subject.Examples of immunotherapeutic agents contemplated for use in combinationwith at least one glutamine antagonist described herein include, but arenot limited to, immune checkpoint molecules (e.g., antibodies to immunecheckpoint proteins), interleukins (e.g., IL-2, IL-7, IL-12, IL-15),cytokines (e.g., interferons, G-CSF, imiquimod), chemokines (e.g., CCL3,CCL26, CXCL7), vaccines (e.g., peptide vaccines, dendritic cell (DC)vaccines, EGFRvIII vaccines, mesothilin vaccine, G-VAX, listeriavaccines), and adoptive T cell therapy including chimeric antigenreceptor T cells (CAR T cells).

As used herein, “anti-inflammatory agent” refers to an agent that may beused to prevent or reduce an inflammatory response or inflammation in acell, tissue, organ, or subject. Exemplary anti-inflammatory agentscontemplated for use include, without limitation, steroidalanti-inflammatory agents, a nonsteroidal anti-inflammatory agent, or acombination thereof. In some embodiments, anti-inflammatory agentsinclude clobetasol, alclofenac, alclometasone dipropionate, algestoneacetonide, alpha amylase, amcinafal, amcinafide, amfenac sodium,amiprilose hydrochloride, anakinra, anirolac, anitrazafen, apazone,balsalazide disodium, bendazac, benoxaprofen, benzydamine hydrochloride,bromelains, broperamole, budesonide, carprofen, cicloprofen, cintazone,cliprofen, clobetasol propionate, clobetasone butyrate, clopirac,cloticasone propionate, cormethasone acetate, cortodoxone, deflazacort,desonide, desoximetasone, dexamethasone, dexamethasone acetate,dexamethasone dipropionate, diclofenac potassium, diclofenac sodium,diflorasone diacetate, diflumidone sodium, diflunisal, difluprednate,diftalone, dimethyl sulfoxide, drocinonide, endrysone, enlimomab,enolicam sodium, epirizole, etodolac, etofenamate, felbinac, fenamole,fenbufen, fenclofenac, fenclorac, fendosal, fenpipalone, fentiazac,flazalone, fluazacort, flufenamic acid, flumizole, flunisolide acetate,flunixin, flunixin meglumine, fluocortin butyl, fluorometholone acetate,fluquazone, flurbiprofen, fluretofen, fluticasone propionate,furaprofen, furobufen, halcinonide, halobetasol propionate, halopredoneacetate, ibufenac, ibuprofen, ibuprofen aluminum, ibuprofen piconol,ilonidap, indomethacin, indomethacin sodium, indoprofen, indoxole,intrazole, isoflupredone acetate, isoxepac, isoxicam, ketoprofen,lofemizole hydrochloride, lomoxicam, loteprednol etabonate,meclofenamate sodium, meclofenamic acid, meclorisone dibutyrate,mefenamic acid, mesalamine, meseclazone, methylprednisolone suleptanate,momiflumate, nabumetone, naproxen, naproxen sodium, naproxol, nimazone,olsalazine sodium, orgotein, orpanoxin, oxaprozin, oxyphenbutazone,paranyline hydrochloride, pentosan polysulfate sodium, phenbutazonesodium glycerate, pirfenidone, piroxicam, piroxicam cinnamate, piroxicamolamine, pirprofen, prednazate, prifelone, prodolic acid, proquazone,proxazole, proxazole citrate, rimexolone, romazarit, salcolex,salnacedin, salsalate, sanguinarium chloride, seclazone, sermetacin,sudoxicam, sulindac, suprofen, talmetacin, talniflumate, talosalate,tebufelone, tenidap, tenidap sodium, tenoxicam, tesicam, tesimide,tetrydamine, tiopinac, tixocortol pivalate, tolmetin, tolmetin sodium,triclonide, triflumidate, zidometacin, zomepirac sodium, aspirin(acetylsalicylic acid), salicylic acid, corticosteroids,glucocorticoids, tacrolimus, pimecorlimus, prodrugs thereof, co-drugsthereof, and combinations thereof. The anti-inflammatory agent may alsobe a biological inhibitor of proinflammatory signaling moleculesincluding antibodies to such biological inflammatory signalingmolecules.

Exemplary neuroprotective agents include, without limitation, L-dopa,dopamine agonists (e.g., apomorphine, bromocriptine, pergolide,ropinirole, pramipexole, or cabergoline), adenosine A2a antagonists(Shah et al., Curr. Opin. Drug Discov. Devel. 13:466-80 (2010));serotonin receptor agonists; continuous-release levodopa (Sinemet CR®,MSD, Israel); continuous duodenal levodopa administration (Duodopa®,Abbott, UK); catechol-O-methyltransferase (COMT) inhibitors (e.g.,Stalevo®, Novartis Pharma, USA; entacapone (Comtan®, Novartis Pharma,USA)); tolcapone; coenzyme Q10, and/or MAO-B inhibitors (e.g.,Selegiline or Rasagiline). Additional neuroprotective agents aredescribed in, e.g., Hart et al., Mov. Disord. 24: 647-54 (2009).

In some contexts, an agent described herein can be administered with anantigen (e.g., to induce an immune response). In some embodiments, anadjuvant can be used in combination with the antigen.

An agent described herein can also be used in combination with animaging agent. An agent (e.g., a glutamine antagonist) can be attachedto imaging agents for imaging and diagnosis of various diseased organs,tissues or cell types. The agent can be labeled or conjugated afluorophore or radiotracer for use as an imaging agent. Many appropriateimaging agents are known in the art, as are methods for their attachmentto agents (e.g., attaching an imaging agent to a proteins or peptidesusing metal chelate complexes, radioisotopes, fluorescent markers, orenzymes whose presence can be detected using a colorimetric markers(such as, but not limited to, urease, alkaline phosphatase,(horseradish) hydrogen peroxidase and glucose oxidase)). An agent mayalso be dual labeled with a radioisotope in order to combine imagingthrough nuclear approaches and be made into a unique cyclic structureand optimized for binding affinity and pharmacokinetics. Such agents canbe administered by any number of methods known to those of ordinaryskill in the art including, but not limited to, oral administration,inhalation, subcutaneous (sub-q), intravenous (I.V.), intraperitoneal(LP.), intramuscular (I.M.), or intrathecal injection.

The presently disclosed subject matter contemplates the use of at leastone glutamine antagonists, alone, or optionally together with one ormore additional therapeutic agents described herein. Accordingly, in anaspect the presently disclosed subject matter involves the use of atleast one glutamine antagonist for treating a cognitive deficit.

II. Pharmaceutical Compositions Comprising Glutamine Antagonists

The presently disclosed subject matter also contemplates pharmaceuticalcompositions comprising one or more glutamine antagonists for thetreatment of a cognitive deficit. In some embodiments, the presentlydisclosed methods comprise the use of the presently disclosed glutamineantagonists for the manufacture of a medicament for the treatment of acognitive deficit.

Accordingly, in some embodiments, the presently disclosed subject matterprovides a pharmaceutical composition comprising an effective amount ofat least one glutamine antagonist that treats a cognitive deficit, and apharmaceutically acceptable carrier, diluent, or excipient.

In some embodiments, the glutamine antagonist composition comprises oneor more additional therapeutic agents described herein (e.g., antiviralagents, immunotherapeutic agents, anti-inflammatory agents,neuroprotective agents, neuroregenerative agents, neurotrophic factors,stem and progenitor cells used to replace and/or repair endogenouspopulations of abnormal, harmful, or unhealthy cells, and vaccines).Generally, the presently disclosed compositions (e.g., comprising atleast one glutamine antagonist) can be administered to a subject fortherapy by any suitable route of administration, including orally,nasally, transmucosally, ocularly, rectally, intravaginally,parenterally, including intramuscular, subcutaneous, intramedullaryinjections, as well as intrathecal, direct intraventricular,intravenous, intra-articular, intra-sternal, intra-synovial,intra-hepatic, intralesional, intracranial, intraperitoneal, intranasal,or intraocular injections, intracisternally, topically, as by powders,ointments or drops (including eyedrops), including buccally andsublingually, transdermally, through an inhalation spray, or other modesof delivery known in the art.

The phrases “systemic administration,” “administered systemically,”“peripheral administration” and “administered peripherally” as usedherein mean the administration of compositions comprising at least oneglutamine antagonist, such that it enters the patient's system and,thus, are subject to metabolism and other like processes, for example,subcutaneous administration.

The phrases “parenteral administration” and “administered parenterally”as used herein mean modes of administration other than enteral andtopical administration, usually by injection, and includes, withoutlimitation, intravenous, intramuscular, intarterial, intrathecal,intracapsular, intraorbital, intraocular, intracardiac, intradermal,intraperitoneal, transtracheal, subcutaneous, subcuticular,intraarticular, subcapsular, subarachnoid, intraspinal and intrasternalinjection and infusion.

The presently disclosed pharmaceutical compositions can be manufacturedin a manner known in the art, e.g., by means of conventional mixing,dissolving, granulating, dragee-making, levitating, emulsifying,encapsulating, entrapping or lyophilizing processes.

In some embodiments, the presently disclosed pharmaceutical compositionscan be administered by rechargeable or biodegradable devices. Forexample, a variety of slow-release polymeric devices have been developedand tested in vivo for the controlled delivery of drugs, includingproteinaceous biopharmaceuticals. Suitable examples of sustained releasepreparations include semipermeable polymer matrices in the form ofshaped articles, e.g., films or microcapsules. Sustained releasematrices include polyesters, hydrogels, polylactides (U.S. Pat. No.3,773,919; EP 58,481), copolymers of L-glutamic acid and gammaethyl-L-glutamate (Sidman et al., Biopolymers 22:547, 1983), poly(2-hydroxyethyl-methacrylate) (Langer et al. (1981) J. Biomed. Mater.Res. 15:167; Langer (1982), Chem. Tech. 12:98), ethylene vinyl acetate(Langer et al. (1981) J. Biomed. Mater. Res. 15:167), orpoly-D-(−)-3-hydroxybutyric acid (EP 133,988A). Sustained releasecompositions also include liposomally entrapped compositions comprisingat least one glutamine antagonist which can be prepared by methods knownin the art (Epstein et al. (1985) Proc. Natl. Acad. Sci. U.S.A. 82:3688;Hwang et al. (1980) Proc. Natl. Acad. Sci. U.S.A. 77:4030; U.S. Pat.Nos. 4,485,045 and 4,544,545; and EP 102,324A). Ordinarily, theliposomes are of the small (about 200-800 angstroms) unilamelar type inwhich the lipid content is greater than about 30 mol % cholesterol, theselected proportion being adjusted for the optimal therapy. Suchmaterials can comprise an implant, for example, for sustained release ofthe presently disclosed compositions, which, in some embodiments, can beimplanted at a particular, pre-determined target site.

In another embodiment, the presently disclosed pharmaceuticalcompositions may comprise PEGylated therapeutics (e.g., PEGylatedantibodies). PEGylation is a well established and validated approach forthe modification of a range of antibodies, proteins, and peptides andinvolves the attachment of polyethylene glycol (PEG) at specific sitesof the antibodies, proteins, and peptides (Chapman (2002) Adv. DrugDeliv. Rev. 54:531-545). Some effects of PEGylation include: (a)markedly improved circulating half-lives in vivo due to either evasionof renal clearance as a result of the polymer increasing the apparentsize of the molecule to above the glomerular filtration limit, and/orthrough evasion of cellular clearance mechanisms; (b) improvedpharmacokinetics; (c) improved solubility—PEG has been found to besoluble in many different solvents, ranging from water to many organicsolvents, such as toluene, methylene chloride, ethanol and acetone; (d)PEGylated antibody fragments can be concentrated to 200 mg/mL, and theability to do so opens up formulation and dosing options, such assubcutaneous administration of a high protein dose; this is in contrastto many other therapeutic antibodies which are typically administeredintravenously; (e) enhanced proteolytic resistance of the conjugatedprotein (Cunningham-Rundles et. al. (1992) J. Immunol. Meth.152:177-190); (f) improved bioavailability via reduced losses atsubcutaneous injection sites; (g) reduced toxicity has been observed;for agents where toxicity is related to peak plasma level, a flatterpharmacokinetic profile achieved by sub-cutaneous administration ofPEGylated protein is advantageous; proteins that elicit an immuneresponse which has toxicity consequences may also benefit as a result ofPEGylation; and (h) improved thermal and mechanical stability of thePEGylated molecule.

Pharmaceutical compositions for parenteral administration includeaqueous solutions of compositions comprising at least one glutamineantagonist. For injection, the presently disclosed pharmaceuticalcompositions can be formulated in aqueous solutions, for example, insome embodiments, in physiologically compatible buffers, such as Hank'ssolution, Ringer's solution, or physiologically buffered saline. Aqueousinjection suspensions can contain substances that increase the viscosityof the suspension, such as sodium carboxymethyl cellulose, sorbitol, ordextran. Additionally, suspensions of compositions include fatty oils,such as sesame oil, or synthetic fatty acid esters, such as ethyl oleateor triglycerides, or liposomes. Optionally, the suspension also cancontain suitable stabilizers or agents that increase the solubility ofthe compositions comprising at least one glutamine antagonist to allowfor the preparation of highly concentrated solutions.

For nasal or transmucosal administration generally, penetrantsappropriate to the particular barrier to be permeated are used in theformulation. Such penetrants are generally known in the art.

Additional ingredients can be added to compositions for topicaladministration, as long as such ingredients are pharmaceuticallyacceptable and not deleterious to the epithelial cells or theirfunction. Further, such additional ingredients should not adverselyaffect the epithelial penetration efficiency of the composition, andshould not cause deterioration in the stability of the composition. Forexample, fragrances, opacifiers, antioxidants, gelling agents,stabilizers, surfactants, emollients, coloring agents, preservatives,buffering agents, and the like can be present. The pH of the presentlydisclosed topical composition can be adjusted to a physiologicallyacceptable range of from about 6.0 to about 9.0 by adding bufferingagents thereto such that the composition is physiologically compatiblewith a subject's skin.

Regardless of the route of administration selected, the presentlydisclosed compositions are formulated into pharmaceutically acceptabledosage forms, such as described herein or by other conventional methodsknown to those of skill in the art.

In general, the “effective amount”, “amount effective to treat” or“therapeutically effective amount” of an active agent or drug deliverydevice refers to the amount necessary to elicit the desired biologicalresponse. As will be appreciated by those of ordinary skill in this art,the effective amount of an agent or device may vary depending on suchfactors as the desired biological endpoint, the agent to be delivered,the composition of the encapsulating matrix, the target tissue, and thelike. Generally, the “effective amount” or “amount effective” to treat acognitive deficit is less than the effective amount needed to treatcancer. It should be appreciated that the amount of at least oneglutamine antagonist, or prodrug or analog thereof, effective to treat acognitive deficit comprises the maximal non-toxic dose that sufficientfor improving a particular cognitive deficit in a subject. In someembodiments, the effective amount of at least one glutamine antagonist,or prodrug or analog thereof, is less than 0.1 mg/kg/day.

The term “combination” is used in its broadest sense and means that asubject is administered at least two agents. More particularly, the term“in combination” refers to the concomitant administration of two (ormore) active agents for the treatment of a, e.g., single disease state.As used herein, the active agents may be combined and administered in asingle dosage form, may be administered as separate dosage forms at thesame time, or may be administered as separate dosage forms that areadministered alternately or sequentially on the same or separate days.In one embodiment of the presently disclosed subject matter, the activeagents are combined and administered in a single dosage form. In anotherembodiment, the active agents are administered in separate dosage forms(e.g., wherein it is desirable to vary the amount of one but not theother). The single dosage form may include additional active agents forthe treatment of the disease state.

Further, the presently disclosed compositions can be administered aloneor in combination with adjuvants that enhance stability of the agents,facilitate administration of pharmaceutical compositions containing themin certain embodiments, provide increased dissolution or dispersion,increase activity, provide adjuvant therapy, and the like, includingother active ingredients. Advantageously, such combination therapiesutilize lower dosages of the conventional therapeutics, thus avoidingpossible toxicity and adverse side effects incurred when those agentsare used as monotherapies.

The timing of administration of at least one glutamine antagonist can bevaried so long as the beneficial effects of the combination of theseagents are achieved. Accordingly, the phrase “in combination with”refers to the administration of at least one glutamine antagonist, andoptionally additional agents either simultaneously, sequentially, or acombination thereof. Therefore, a subject administered a combination ofat least one glutamine antagonist, and optionally additional agents canreceive at least one glutamine antagonist, and optionally additionalagents at the same time (i.e., simultaneously) or at different times(i.e., sequentially, in either order, on the same day or on differentdays), so long as the effect of the combination of all agents isachieved in the subject.

When administered sequentially, the agents can be administered within 1,5, 10, 30, 60, 120, 180, 240 minutes or longer of one another. In otherembodiments, agents administered sequentially, can be administeredwithin 1, 2, 3, 4, 5, 10, 15, 20 or more days of one another. Where theagents are administered simultaneously, they can be administered to thesubject as separate pharmaceutical compositions, each comprising atleast one glutamine antagonist, and optionally additional agents, orthey can be administered to a subject as a single pharmaceuticalcomposition comprising all agents.

When administered in combination, the effective concentration of each ofthe agents to elicit a particular biological response may be less thanthe effective concentration of each agent when administered alone,thereby allowing a reduction in the dose of one or more of the agentsrelative to the dose that would be needed if the agent was administeredas a single agent. The effects of multiple agents may, but need not be,additive or synergistic. The agents may be administered multiple times.

In some embodiments, when administered in combination, the two or moreagents can have a synergistic effect. As used herein, the terms“synergy,” “synergistic,” “synergistically” and derivations thereof,such as in a “synergistic effect” or a “synergistic combination” or a“synergistic composition” refer to circumstances under which thebiological activity of a combination of an agent and at least oneadditional therapeutic agent is greater than the sum of the biologicalactivities of the respective agents when administered individually.

Synergy can be expressed in terms of a “Synergy Index (SI),” whichgenerally can be determined by the method described by F. C. Kull et al.Applied Microbiology 9, 538 (1961), from the ratio determined by:

Q _(a) Q _(A) +Q _(b) Q _(B)=Synergy Index (SI)

wherein:

-   -   Q_(A) is the concentration of a component A, acting alone, which        produced an end point in relation to component A;    -   Q_(a) is the concentration of component A, in a mixture, which        produced an end point;    -   Q_(B) is the concentration of a component B, acting alone, which        produced an end point in relation to component B; and    -   Q_(b) is the concentration of component B, in a mixture, which        produced an end point.

Generally, when the sum of Q_(a)/Q_(A) and Q_(b)/Q_(B) is greater thanone, antagonism is indicated. When the sum is equal to one, additivityis indicated. When the sum is less than one, synergism is demonstrated.The lower the SI, the greater the synergy shown by that particularmixture. Thus, a “synergistic combination” has an activity higher thatwhat can be expected based on the observed activities of the individualcomponents when used alone. Further, a “synergistically effectiveamount” of a component refers to the amount of the component necessaryto elicit a synergistic effect in, for example, another therapeuticagent present in the composition.

In another aspect, the presently disclosed subject matter provides apharmaceutical composition including at least one glutamine antagonist,and optionally additional agents, alone or in combination with one ormore additional therapeutic agents in admixture with a pharmaceuticallyacceptable excipient.

More particularly, the presently disclosed subject matter provides apharmaceutical composition comprising at least one glutamine antagonist,and optionally additional agents, and a pharmaceutically acceptablecarrier. In therapeutic and/or diagnostic applications, the compounds ofthe disclosure can be formulated for a variety of modes ofadministration, including systemic and topical or localizedadministration. Techniques and formulations generally may be found inRemington: The Science and Practice of Pharmacy (20^(th) ed.)Lippincott, Williams and Wilkins (2000).

Use of pharmaceutically acceptable inert carriers to formulate thecompounds herein disclosed for the practice of the disclosure intodosages suitable for systemic administration is within the scope of thedisclosure. With proper choice of carrier and suitable manufacturingpractice, the compositions of the present disclosure, in particular,those formulated as solutions, may be administered parenterally, such asby intravenous injection. The compounds can be formulated readily usingpharmaceutically acceptable carriers well known in the art into dosagessuitable for oral administration. Such carriers enable the compounds ofthe disclosure to be formulated as tablets, pills, capsules, liquids,gels, syrups, slurries, suspensions and the like, for oral ingestion bya subject (e.g., patient) to be treated.

For nasal or inhalation delivery, the agents of the disclosure also maybe formulated by methods known to those of skill in the art, and mayinclude, for example, but not limited to, examples of solubilizing,diluting, or dispersing substances, such as saline; preservatives, suchas benzyl alcohol; absorption promoters; and fluorocarbons.

Pharmaceutical compositions suitable for use in the present disclosureinclude compositions wherein the active ingredients are contained in aneffective amount to achieve its intended purpose. Determination of theeffective amounts is well within the capability of those skilled in theart, especially in light of the detailed disclosure provided herein.Generally, the compounds according to the disclosure are effective overa wide dosage range. For example, in the treatment of adult humans,dosages from 0.01 to 1000 mg, from 0.5 to 100 mg, from 1 to 50 mg perday, and from 5 to 40 mg per day are examples of dosages that may beused. A non-limiting dosage is 10 to 30 mg per day. The exact dosagewill depend upon the route of administration, the form in which thecompound is administered, the subject to be treated, the body weight ofthe subject to be treated, and the preference and experience of theattending physician.

In addition to the active ingredients, these pharmaceutical compositionsmay contain suitable pharmaceutically acceptable carriers comprisingexcipients and auxiliaries which facilitate processing of the activecompounds into preparations which can be used pharmaceutically. Thepreparations formulated for oral administration may be in the form oftablets, dragees, capsules, or solutions.

III. General Definitions

Although specific terms are employed herein, they are used in a genericand descriptive sense only and not for purposes of limitation. Unlessotherwise defined, all technical and scientific terms used herein havethe same meaning as commonly understood by one of ordinary skill in theart to which this presently described subject matter belongs.

While the following terms in relation to compounds of Formula (I) arebelieved to be well understood by one of ordinary skill in the art, thefollowing definitions are set forth to facilitate explanation of thepresently disclosed subject matter. These definitions are intended tosupplement and illustrate, not preclude, the definitions that would beapparent to one of ordinary skill in the art upon review of the presentdisclosure.

The terms substituted, whether preceded by the term “optionally” or not,and substituent, as used herein, refer to the ability, as appreciated byone skilled in this art, to change one functional group for anotherfunctional group on a molecule, provided that the valency of all atomsis maintained. When more than one position in any given structure may besubstituted with more than one substituent selected from a specifiedgroup, the substituent may be either the same or different at everyposition. The substituents also may be further substituted (e.g., anaryl group substituent may have another substituent off it, such asanother aryl group, which is further substituted at one or morepositions).

Where substituent groups or linking groups are specified by theirconventional chemical formulae, written from left to right, they equallyencompass the chemically identical substituents that would result fromwriting the structure from right to left, e.g., —CH₂O—is equivalent to—OCH₂—; —C(═O)O— is equivalent to —OC(═O)—; —OC(═O)NR— is equivalent to—NRC(═O)O—, and the like.

When the term “independently selected” is used, the substituents beingreferred to (e.g., R groups, such as groups R₁, R₂, and the like, orvariables, such as “m” and “n”), can be identical or different. Forexample, both R₁ and R₂ can be substituted alkyls, or R₁ can be hydrogenand R₂ can be a substituted alkyl, and the like.

The terms “a,” “an,” or “a(n),” when used in reference to a group ofsubstituents herein, mean at least one. For example, where a compound issubstituted with “an” alkyl or aryl, the compound is optionallysubstituted with at least one alkyl and/or at least one aryl. Moreover,where a moiety is substituted with an R substituent, the group may bereferred to as “R-substituted.” Where a moiety is R-substituted, themoiety is substituted with at least one R substituent and each Rsubstituent is optionally different.

A named “R” or group will generally have the structure that isrecognized in the art as corresponding to a group having that name,unless specified otherwise herein. For the purposes of illustration,certain representative “R” groups as set forth above are defined below.

Descriptions of compounds of the present disclosure are limited byprinciples of chemical bonding known to those skilled in the art.Accordingly, where a group may be substituted by one or more of a numberof substituents, such substitutions are selected so as to comply withprinciples of chemical bonding and to give compounds which are notinherently unstable and/or would be known to one of ordinary skill inthe art as likely to be unstable under ambient conditions, such asaqueous, neutral, and several known physiological conditions. Forexample, a heterocycloalkyl or heteroaryl is attached to the remainderof the molecule via a ring heteroatom in compliance with principles ofchemical bonding known to those skilled in the art thereby avoidinginherently unstable compounds.

Unless otherwise explicitly defined, a “substituent group,” as usedherein, includes a functional group selected from one or more of thefollowing moieties, which are defined herein:

The term hydrocarbon, as used herein, refers to any chemical groupcomprising hydrogen and carbon. The hydrocarbon may be substituted orunsubstituted. As would be known to one skilled in this art, allvalencies must be satisfied in making any substitutions. The hydrocarbonmay be unsaturated, saturated, branched, unbranched, cyclic, polycyclic,or heterocyclic. Illustrative hydrocarbons are further defined hereinbelow and include, for example, methyl, ethyl, n-propyl, isopropyl,cyclopropyl, allyl, vinyl, n-butyl, tert-butyl, ethynyl, cyclohexyl, andthe like.

The term “alkyl,” by itself or as part of another substituent, means,unless otherwise stated, a straight (i.e., unbranched) or branchedchain, acyclic or cyclic hydrocarbon group, or combination thereof,which may be fully saturated, mono- or polyunsaturated and can includedi- and multivalent groups, having the number of carbon atoms designated(i.e., C₁-C₁₀ means one to ten carbons, including 1, 2, 3, 4, 5, 6, 7,8, 9, and 10 carbons). In particular embodiments, the term “alkyl”refers to C₁₋₂₀ inclusive, including 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, and 20 carbons, linear (i.e.,“straight-chain”), branched, or cyclic, saturated or at least partiallyand in some cases fully unsaturated (i.e., alkenyl and alkynyl)hydrocarbon radicals derived from a hydrocarbon moiety containingbetween one and twenty carbon atoms by removal of a single hydrogenatom.

Representative saturated hydrocarbon groups include, but are not limitedto, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl,tert-butyl, n-pentyl, sec-pentyl, isopentyl, neopentyl, n-hexyl,sec-hexyl, n-heptyl, n-octyl, n-decyl, n-undecyl, dodecyl, cyclohexyl,(cyclohexyl)methyl, cyclopropylmethyl, and homologs and isomers thereof.

“Branched” refers to an alkyl group in which a lower alkyl group, suchas methyl, ethyl or propyl, is attached to a linear alkyl chain. “Loweralkyl” refers to an alkyl group having 1 to about 8 carbon atoms (i.e.,a C₁₋₈ alkyl), e.g., 1, 2, 3, 4, 5, 6, 7, or 8 carbon atoms. “Higheralkyl” refers to an alkyl group having about 10 to about 20 carbonatoms, e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 carbon atoms.In certain embodiments, “alkyl” refers, in particular, to C₁₋₈straight-chain alkyls. In other embodiments, “alkyl” refers, inparticular, to C₁₋₈ branched-chain alkyls.

Alkyl groups can optionally be substituted (a “substituted alkyl”) withone or more alkyl group substituents, which can be the same ordifferent. The term “alkyl group substituent” includes but is notlimited to alkyl, substituted alkyl, halo, arylamino, acyl, hydroxyl,aryloxyl, alkoxyl, alkylthio, arylthio, aralkyloxyl, aralkylthio,carboxyl, alkoxycarbonyl, oxo, and cycloalkyl. There can be optionallyinserted along the alkyl chain one or more oxygen, sulfur or substitutedor unsubstituted nitrogen atoms, wherein the nitrogen substituent ishydrogen, lower alkyl (also referred to herein as “alkylaminoalkyl”), oraryl.

Thus, as used herein, the term “substituted alkyl” includes alkylgroups, as defined herein, in which one or more atoms or functionalgroups of the alkyl group are replaced with another atom or functionalgroup, including for example, alkyl, substituted alkyl, halogen, aryl,substituted aryl, alkoxyl, hydroxyl, nitro, amino, alkylamino,dialkylamino, sulfate, and mercapto.

The term “heteroalkyl,” by itself or in combination with another term,means, unless otherwise stated, a stable straight or branched chain, orcyclic hydrocarbon group, or combinations thereof, consisting of atleast one carbon atoms and at least one heteroatom selected from thegroup consisting of O, N, P, Si and S, and wherein the nitrogen,phosphorus, and sulfur atoms may optionally be oxidized and the nitrogenheteroatom may optionally be quaternized. The heteroatom(s) O, N, P andS and Si may be placed at any interior position of the heteroalkyl groupor at the position at which alkyl group is attached to the remainder ofthe molecule. Examples include, but are not limited to, —CH₂—CH₂—O—CH₃,—CH₂—CH₂—NH—CH₃, —CH₂—CH₂—N(CH₃)—CH₃, —CH₂—S—CH₂—CH₃,—CH₂—CH₂₅—S(O)—CH₃, —CH₂—CH₂—S(O)₂—CH₃, —CH═CHO—CH₃, —Si(CH₃)₃,—CH₂—CH═N—OCH₃, —CH═CH—N(CH₃)—CH₃, O—CH₃, —O—CH₂—CH₃, and —CN. Up to twoor three heteroatoms may be consecutive, such as, for example,—CH₂—NH—OCH₃ and —CH₂—O—Si(CH₃)₃.

As described above, heteroalkyl groups, as used herein, include thosegroups that are attached to the remainder of the molecule through aheteroatom, such as —C(O)NR′, —NR′R″, —OR′, —SR, —S(O)R, and/or—S(O₂)R′. Where “heteroalkyl” is recited, followed by recitations ofspecific heteroalkyl groups, such as —NR′R or the like, it will beunderstood that the terms heteroalkyl and —NR′R″ are not redundant ormutually exclusive. Rather, the specific heteroalkyl groups are recitedto add clarity. Thus, the term “heteroalkyl” should not be interpretedherein as excluding specific heteroalkyl groups, such as —NR′R″ or thelike.

“Cyclic” and “cycloalkyl” refer to a non-aromatic mono- or multicyclicring system of about 3 to about 10 carbon atoms, e.g., 3, 4, 5, 6, 7, 8,9, or 10 carbon atoms. The cycloalkyl group can be optionally partiallyunsaturated. The cycloalkyl group also can be optionally substitutedwith an alkyl group substituent as defined herein, oxo, and/or alkylene.There can be optionally inserted along the cyclic alkyl chain one ormore oxygen, sulfur or substituted or unsubstituted nitrogen atoms,wherein the nitrogen substituent is hydrogen, unsubstituted alkyl,substituted alkyl, aryl, or substituted aryl, thus providing aheterocyclic group. Representative monocyclic cycloalkyl rings includecyclopentyl, cyclohexyl, and cycloheptyl. Multicyclic cycloalkyl ringsinclude adamantyl, octahydronaphthyl, decalin, camphor, camphane, andnoradamantyl, and fused ring systems, such as dihydro- andtetrahydronaphthalene, and the like.

The term “cycloalkylalkyl,” as used herein, refers to a cycloalkyl groupas defined hereinabove, which is attached to the parent molecular moietythrough an alkyl group, also as defined above. Examples ofcycloalkylalkyl groups include cyclopropylmethyl and cyclopentylethyl.

The terms “cycloheteroalkyl” or “heterocycloalkyl” refer to anon-aromatic ring system, unsaturated or partially unsaturated ringsystem, such as a 3- to 10-member substituted or unsubstitutedcycloalkyl ring system, including one or more heteroatoms, which can bethe same or different, and are selected from the group consisting ofnitrogen (N), oxygen (O), sulfur (S), phosphorus (P), and silicon (Si),and optionally can include one or more double bonds.

The cycloheteroalkyl ring can be optionally fused to or otherwiseattached to other cycloheteroalkyl rings and/or non-aromatic hydrocarbonrings. Heterocyclic rings include those having from one to threeheteroatoms independently selected from oxygen, sulfur, and nitrogen, inwhich the nitrogen and sulfur heteroatoms may optionally be oxidized andthe nitrogen heteroatom may optionally be quaternized. In certainembodiments, the term heterocylic refers to a non-aromatic 5-, 6-, or7-membered ring or a polycyclic group wherein at least one ring atom isa heteroatom selected from O, S, and N (wherein the nitrogen and sulfurheteroatoms may be optionally oxidized), including, but not limited to,a bi- or tri-cyclic group, comprising fused six-membered rings havingbetween one and three heteroatoms independently selected from theoxygen, sulfur, and nitrogen, wherein (i) each 5-membered ring has 0 to2 double bonds, each 6-membered ring has 0 to 2 double bonds, and each7-membered ring has 0 to 3 double bonds, (ii) the nitrogen and sulfurheteroatoms may be optionally oxidized, (iii) the nitrogen heteroatommay optionally be quaternized, and (iv) any of the above heterocyclicrings may be fused to an aryl or heteroaryl ring. Representativecycloheteroalkyl ring systems include, but are not limited topyrrolidinyl, pyrrolinyl, imidazolidinyl, imidazolinyl, pyrazolidinyl,pyrazolinyl, piperidyl, piperazinyl, indolinyl, quinuclidinyl,morpholinyl, thiomorpholinyl, thiadiazinanyl, tetrahydrofuranyl, and thelike.

The terms “cycloalkyl” and “heterocycloalkyl”, by themselves or incombination with other terms, represent, unless otherwise stated, cyclicversions of “alkyl” and “heteroalkyl”, respectively. Additionally, forheterocycloalkyl, a heteroatom can occupy the position at which theheterocycle is attached to the remainder of the molecule. Examples ofcycloalkyl include, but are not limited to, cyclopentyl, cyclohexyl,1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl, and the like. Examples ofheterocycloalkyl include, but are not limited to,1-(1,2,5,6-tetrahydropyridyl), 1-piperidinyl, 2-piperidinyl,3-piperidinyl, 4-morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl,tetrahydrofuran-3-yl, tetrahydrothien-2-yl, tetrahydrothien-3-yl,1-piperazinyl, 2-piperazinyl, and the like. The terms “cycloalkylene”and “heterocycloalkylene” refer to the divalent derivatives ofcycloalkyl and heterocycloalkyl, respectively.

An unsaturated alkyl group is one having one or more double bonds ortriple bonds. Examples of unsaturated alkyl groups include, but are notlimited to, vinyl, 2-propenyl, crotyl, 2-isopentenyl, 2-(butadienyl),2,4-pentadienyl, 3-(1,4-pentadienyl), ethynyl, 1- and 3-propynyl,3-butynyl, and the higher homologs and isomers. Alkyl groups which arelimited to hydrocarbon groups are termed “homoalkyl.”

More particularly, the term “alkenyl” as used herein refers to amonovalent group derived from a C₁₋₂₀ inclusive straight or branchedhydrocarbon moiety having at least one carbon-carbon double bond by theremoval of a single hydrogen molecule. Alkenyl groups include, forexample, ethenyl (i.e., vinyl), propenyl, butenyl,1-methyl-2-buten-1-yl, pentenyl, hexenyl, octenyl, allenyl, andbutadienyl.

The term “cycloalkenyl” as used herein refers to a cyclic hydrocarboncontaining at least one carbon-carbon double bond. Examples ofcycloalkenyl groups include cyclopropenyl, cyclobutenyl, cyclopentenyl,cyclopentadiene, cyclohexenyl, 1,3-cyclohexadiene, cycloheptenyl,cycloheptatrienyl, and cyclooctenyl.

The term “alkynyl” as used herein refers to a monovalent group derivedfrom a straight or branched C₁₋₂₀ hydrocarbon of a designed number ofcarbon atoms containing at least one carbon-carbon triple bond. Examplesof “alkynyl” include ethynyl, 2-propynyl (propargyl), 1-propynyl,pentynyl, hexynyl, and heptynyl groups, and the like.

The term “alkylene” by itself or a part of another substituent refers toa straight or branched bivalent aliphatic hydrocarbon group derived froman alkyl group having from 1 to about 20 carbon atoms, e.g., 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 carbonatoms. The alkylene group can be straight, branched or cyclic. Thealkylene group also can be optionally unsaturated and/or substitutedwith one or more “alkyl group substituents.” There can be optionallyinserted along the alkylene group one or more oxygen, sulfur orsubstituted or unsubstituted nitrogen atoms (also referred to herein as“alkylaminoalkyl”), wherein the nitrogen substituent is alkyl aspreviously described. Exemplary alkylene groups include methylene(—CH₂—); ethylene (—CH₂—CH₂—); propylene (—(CH₂)₃—); cyclohexylene(—C₆H₁₀—); —CH═CH—CH═CH—; —CH═CH—CH₂—; —CH₂CH₂CH₂CH₂—, —CH₂CH═CHCH₂—,—CH₂CsCCH₂—, —CH₂CH₂CH(CH₂CH₂CH₃)CH₂—, —(CH₂)_(q)—N(R)—(CH₂)_(r)—,wherein each of q and r is independently an integer from 0 to about 20,e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11. 12, 13, 14, 15, 16, 17, 18,19, or 20, and R is hydrogen or lower alkyl; methylenedioxyl(—O—CH₂—O—); and ethylenedioxyl (—O—(CH₂)₂—O—). An alkylene group canhave about 2 to about 3 carbon atoms and can further have 6-20 carbons.Typically, an alkyl (or alkylene) group will have from 1 to 24 carbonatoms, with those groups having 10 or fewer carbon atoms being someembodiments of the present disclosure. A “lower alkyl” or “loweralkylene” is a shorter chain alkyl or alkylene group, generally havingeight or fewer carbon atoms.

The term “heteroalkylene” by itself or as part of another substituentmeans a divalent group derived from heteroalkyl, as exemplified, but notlimited by, —CH₂—CH₂—S—CH₂—CH₂— and —CH₂—S—CH₂—CH₂—NH—CH₂—. Forheteroalkylene groups, heteroatoms also can occupy either or both of thechain termini (e.g., alkyleneoxo, alkylenedioxo, alkyleneamino,alkylenediamino, and the like). Still further, for alkylene andheteroalkylene linking groups, no orientation of the linking group isimplied by the direction in which the formula of the linking group iswritten. For example, the formula —C(O)OR′— represents both —C(O)OR′—and —R′OC(O)—.

The term “aryl” means, unless otherwise stated, an aromatic hydrocarbonsubstituent that can be a single ring or multiple rings (such as from 1to 3 rings), which are fused together or linked covalently. The term“heteroaryl” refers to aryl groups (or rings) that contain from one tofour heteroatoms (in each separate ring in the case of multiple rings)selected from N, O, and S, wherein the nitrogen and sulfur atoms areoptionally oxidized, and the nitrogen atom(s) are optionallyquaternized. A heteroaryl group can be attached to the remainder of themolecule through a carbon or heteroatom. Non-limiting examples of aryland heteroaryl groups include phenyl, 1-naphthyl, 2-naphthyl,4-biphenyl, 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 3-pyrazolyl,2-imidazolyl, 4-imidazolyl, pyrazinyl, 2-oxazolyl, 4-oxazolyl,2-phenyl-4-oxazolyl, 5-oxazolyl, 3-isoxazolyl, 4-isoxazolyl,5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl,2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl,4-pyrimidyl, 5-benzothiazolyl, purinyl, 2-benzimidazolyl, 5-indolyl,1-isoquinolyl, 5-isoquinolyl, 2-quinoxalinyl, 5-quinoxalinyl,3-quinolyl, and 6-quinolyl. Substituents for each of above noted aryland heteroaryl ring systems are selected from the group of acceptablesubstituents described below. The terms “arylene” and “heteroarylene”refer to the divalent forms of aryl and heteroaryl, respectively.

For brevity, the term “aryl” when used in combination with other terms(e.g., aryloxy, arylthioxy, arylalkyl) includes both aryl and heteroarylrings as defined above. Thus, the terms “arylalkyl” and“heteroarylalkyl” are meant to include those groups in which an aryl orheteroaryl group is attached to an alkyl group (e.g., benzyl, phenethyl,pyridylmethyl, furylmethyl, and the like) including those alkyl groupsin which a carbon atom (e.g., a methylene group) has been replaced by,for example, an oxygen atom (e.g., phenoxymethyl, 2-pyridyloxymethyl,3-(1-naphthyloxy)propyl, and the like). However, the term “haloaryl,” asused herein is meant to cover only aryls substituted with one or morehalogens.

Where a heteroalkyl, heterocycloalkyl, or heteroaryl includes a specificnumber of members (e.g. “3 to 7 membered”), the term “member” refers toa carbon or heteroatom.

Further, a structure represented generally by the formula:

-   -   as used herein refers to a ring structure, for example, but not        limited to a 3-carbon, a 4-carbon, a 5-carbon, a 6-carbon, a        7-carbon, and the like, aliphatic and/or aromatic cyclic        compound, including a saturated ring structure, a partially        saturated ring structure, and an unsaturated ring structure,        comprising a substituent R group, wherein the R group can be        present or absent, and when present, one or more R groups can        each be substituted on one or more available carbon atoms of the        ring structure. The presence or absence of the R group and        number of R groups is determined by the value of the variable        “n,” which is an integer generally having a value ranging from 0        to the number of carbon atoms on the ring available for        substitution. Each R group, if more than one, is substituted on        an available carbon of the ring structure rather than on another        R group. For example, the structure above where n is 0 to 2        would comprise compound groups including, but not limited to:

-   -   and the like.

A dashed line representing a bond in a cyclic ring structure indicatesthat the bond can be either present or absent in the ring. That is, adashed line representing a bond in a cyclic ring structure indicatesthat the ring structure is selected from the group consisting of asaturated ring structure, a partially saturated ring structure, and anunsaturated ring structure.

The symbol (

) denotes the point of attachment of a moiety to the remainder of themolecule.

When a named atom of an aromatic ring or a heterocyclic aromatic ring isdefined as being “absent,” the named atom is replaced by a direct bond.

Each of above terms (e.g., “alkyl,” “heteroalkyl,” “cycloalkyl, and“heterocycloalkyl”, “aryl,” “heteroaryl,” “phosphonate,” and “sulfonate”as well as their divalent derivatives) are meant to include bothsubstituted and unsubstituted forms of the indicated group. Optionalsubstituents for each type of group are provided below.

Substituents for alkyl, heteroalkyl, cycloalkyl, heterocycloalkylmonovalent and divalent derivative groups (including those groups oftenreferred to as alkylene, alkenyl, heteroalkylene, heteroalkenyl,alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, andheterocycloalkenyl) can be one or more of a variety of groups selectedfrom, but not limited to: —OR′, ═O, ═NR′, ═N—OR′, —NR′R″, —SR′,-halogen, —SiR′R″R′″, —OC(O)R′, —C(O)R′, —CO₂R′, —C(O)NR′R″,—OC(O)NR′R″, —NR″C(O)R′, —NR′—C(O)NR″R′, —NR″C(O)OR′, —NR—C(NR′R″)═NR′,—S(O)R′, —S(O)₂R′, —S(O)₂NR′R″, —NRSO₂R′, —CN and —NO₂ in a numberranging from zero to (2m′+1), where m′ is the total number of carbonatoms in such groups. R′, R″, R′″ and R″″ each may independently referto hydrogen, substituted or unsubstituted heteroalkyl, substituted orunsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl,substituted or unsubstituted aryl (e.g., aryl substituted with 1-3halogens), substituted or unsubstituted alkyl, alkoxy or thioalkoxygroups, or arylalkyl groups. As used herein, an “alkoxy” group is analkyl attached to the remainder of the molecule through a divalentoxygen. When a compound of the disclosure includes more than one Rgroup, for example, each of the R groups is independently selected asare each R′, R″, R′″ and R″″ groups when more than one of these groupsis present. When R′ and R″ are attached to the same nitrogen atom, theycan be combined with the nitrogen atom to form a 4-, 5-, 6-, or7-membered ring. For example, —NR′R″ is meant to include, but not belimited to, 1-pyrrolidinyl and 4-morpholinyl. From the above discussionof substituents, one of skill in the art will understand that the term“alkyl” is meant to include groups including carbon atoms bound togroups other than hydrogen groups, such as haloalkyl (e.g., —CF₃ and—CH₂CF₃) and acyl (e.g., —C(O)CH₃, —C(O)CF₃, —C(O)CH₂OCH₃, and thelike).

Similar to the substituents described for alkyl groups above, exemplarysubstituents for aryl and heteroaryl groups (as well as their divalentderivatives) are varied and are selected from, for example: halogen,—OR′, —NR′R″, —SR′, —SiR′R″R″, —OC(O)R′, —C(O)R′, —CO₂R′, —C(O)NR′R″,—OC(O)NR′R″, —NR″C(O)R′, —NR′—C(O)NR″R′″, —NR″C(O)OR′,—NR—C(NR′R″R′)═NR″″, —NR—C(NR′R″)═NR″—S(O)R′, —S(O)₂R′, —S(O)₂NR′R″,—NRSO₂R′, —CN and —NO₂, —R′, —N₃, —CH(Ph)₂, fluoro(C₁-C₄)alkoxo, andfluoro(C₁-C₄)alkyl, in a number ranging from zero to the total number ofopen valences on aromatic ring system; and where R′, R″, R′″ and R″″ maybe independently selected from hydrogen, substituted or unsubstitutedalkyl, substituted or unsubstituted heteroalkyl, substituted orunsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl,substituted or unsubstituted aryl and substituted or unsubstitutedheteroaryl. When a compound of the disclosure includes more than one Rgroup, for example, each of the R groups is independently selected asare each R′, R″, R′ and R″″ groups when more than one of these groups ispresent.

Two of the substituents on adjacent atoms of aryl or heteroaryl ring mayoptionally form a ring of the formula —T—C(O)—(CRR′)_(q)—U—, wherein Tand U are independently —NR—, —O—, —CRR′— or a single bond, and q is aninteger of from 0 to 3. Alternatively, two of the substituents onadjacent atoms of aryl or heteroaryl ring may optionally be replacedwith a substituent of the formula -A-(CH₂)_(r)—B—, wherein A and B areindependently —CRR′—, —O—, —NR—, —S—, —S(O)—, —S(O)₂—, —S(O)₂NR′— or asingle bond, and r is an integer of from 1 to 4.

One of the single bonds of the new ring so formed may optionally bereplaced with a double bond. Alternatively, two of the substituents onadjacent atoms of aryl or heteroaryl ring may optionally be replacedwith a substituent of the formula —(CRR′)_(s)—X′—(C″R′″)_(a)—, where sand d are independently integers of from 0 to 3, and X is —O—, —NR′—,—S—, —S(O)—, —S(O)₂—, or —S(O)₂NR′—. The substituents R, R′, R″ and R′″may be independently selected from hydrogen, substituted orunsubstituted alkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl, and substituted or unsubstituted heteroaryl.

As used herein, the term “acyl” refers to an organic acid group whereinthe —OH of the carboxyl group has been replaced with another substituentand has the general formula RC(═O)—, wherein R is an alkyl, alkenyl,alkynyl, aryl, carbocylic, heterocyclic, or aromatic heterocyclic groupas defined herein). As such, the term “acyl” specifically includesarylacyl groups, such as a 2-(furan-2-yl)acetyl)- and a 2-phenylacetylgroup. Specific examples of acyl groups include acetyl and benzoyl. Acylgroups also are intended to include amides, —RC(═O)NR′, esters,—RC(═O)OR′, ketones, —RC(═O)R′, and aldehydes, —RC(═O)H.

The terms “alkoxyl” or “alkoxy” are used interchangeably herein andrefer to a saturated (i.e., alkyl-O—) or unsaturated (i.e., alkenyl-O—and alkynyl-O—) group attached to the parent molecular moiety through anoxygen atom, wherein the terms “alkyl,” “alkenyl,” and “alkynyl” are aspreviously described and can include C₁₋₂₀ inclusive, linear, branched,or cyclic, saturated or unsaturated oxo-hydrocarbon chains, including,for example, methoxyl, ethoxyl, propoxyl, isopropoxyl, n-butoxyl,sec-butoxyl, tert-butoxyl, and n-pentoxyl, neopentoxyl, n-hexoxyl, andthe like.

The term “alkoxyalkyl” as used herein refers to an alkyl-O-alkyl ether,for example, a methoxyethyl or an ethoxymethyl group.

“Aryloxyl” refers to an aryl-O— group wherein the aryl group is aspreviously described, including a substituted aryl. The term “aryloxyl”as used herein can refer to phenyloxyl or hexyloxyl, and alkyl,substituted alkyl, halo, or alkoxyl substituted phenyloxyl or hexyloxyl.

“Aralkyl” refers to an aryl-alkyl-group wherein aryl and alkyl are aspreviously described, and included substituted aryl and substitutedalkyl. Exemplary aralkyl groups include benzyl, phenylethyl, andnaphthylmethyl.

“Aralkyloxyl” refers to an aralkyl-O— group wherein the aralkyl group isas previously described. An exemplary aralkyloxyl group is benzyloxyl,i.e., C₆H₅—CH₂—O—. An aralkyloxyl group can optionally be substituted.

“Alkoxycarbonyl” refers to an alkyl-O—C(═O)— group. Exemplaryalkoxycarbonyl groups include methoxycarbonyl, ethoxycarbonyl,butyloxycarbonyl, and tert-butyloxycarbonyl.

“Aryloxycarbonyl” refers to an aryl-O—C(═O)— group. Exemplaryaryloxycarbonyl groups include phenoxy- and naphthoxy-carbonyl.

“Aralkoxycarbonyl” refers to an aralkyl-O—C(═O)— group. An exemplaryaralkoxycarbonyl group is benzyloxycarbonyl.

“Carbamoyl” refers to an amide group of the formula —C(═O)NH₂.“Alkylcarbamoyl” refers to a R′RN—C(═O)— group wherein one of R and R′is hydrogen and the other of R and R′ is alkyl and/or substituted alkylas previously described. “Dialkylcarbamoyl” refers to a R′RN—C(═O)—group wherein each of R and R′ is independently alkyl and/or substitutedalkyl as previously described.

The term carbonyldioxyl, as used herein, refers to a carbonate group ofthe formula —O—C(═O)—OR.

“Acyloxyl” refers to an acyl-O— group wherein acyl is as previouslydescribed.

The term “amino” refers to the —NH₂ group and also refers to a nitrogencontaining group as is known in the art derived from ammonia by thereplacement of one or more hydrogen radicals by organic radicals. Forexample, the terms “acylamino” and “alkylamino” refer to specificN-substituted organic radicals with acyl and alkyl substituent groupsrespectively.

An “aminoalkyl” as used herein refers to an amino group covalently boundto an alkylene linker. More particularly, the terms alkylamino,dialkylamino, and trialkylamino as used herein refer to one, two, orthree, respectively, alkyl groups, as previously defined, attached tothe parent molecular moiety through a nitrogen atom. The term alkylaminorefers to a group having the structure —NHR′ wherein R′ is an alkylgroup, as previously defined; whereas the term dialkylamino refers to agroup having the structure —NR′R″, wherein R′ and R″ are eachindependently selected from the group consisting of alkyl groups. Theterm trialkylamino refers to a group having the structure —NR′R″R′″,wherein R′, R″, and R′″ are each independently selected from the groupconsisting of alkyl groups. Additionally, R′, R″, and/or R′″ takentogether may optionally be —(CH₂)_(k)— where k is an integer from 2 to6. Examples include, but are not limited to, methylamino, dimethylamino,ethylamino, diethylamino, diethylaminocarbonyl, methylethylamino,isopropylamino, piperidino, trimethylamino, and propylamino.

The amino group is —NR′R″, wherein R′ and R″ are typically selected fromhydrogen, substituted or unsubstituted alkyl, substituted orunsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl, or substituted or unsubstituted heteroaryl.

The terms alkylthioether and thioalkoxyl refer to a saturated (i.e.,alkyl-S—) or unsaturated (i.e., alkenyl-S— and alkynyl-S—) groupattached to the parent molecular moiety through a sulfur atom. Examplesof thioalkoxyl moieties include, but are not limited to, methylthio,ethylthio, propylthio, isopropylthio, n-butylthio, and the like.

“Acylamino” refers to an acyl-NH— group wherein acyl is as previouslydescribed. “Aroylamino” refers to an aroyl-NH— group wherein aroyl is aspreviously described.

The term “carbonyl” refers to the —C(═O)— group, and can include analdehyde group represented by the general formula R—C(═O)H.

The term “carboxyl” refers to the —COOH group. Such groups also arereferred to herein as a “carboxylic acid” moiety.

The terms “halo,” “halide,” or “halogen” as used herein refer to fluoro,chloro, bromo, and iodo groups. Additionally, terms, such as“haloalkyl,” are meant to include monohaloalkyl and polyhaloalkyl. Forexample, the term “halo(C₁-C₄)alkyl” is mean to include, but not belimited to, trifluoromethyl, 2,2,2-trifluoroethyl, 4-chlorobutyl,3-bromopropyl, and the like.

The term “hydroxyl” refers to the —OH group.

The term “hydroxyalkyl” refers to an alkyl group substituted with an —OHgroup.

The term “mercapto” refers to the —SH group.

The term “oxo” as used herein means an oxygen atom that is double bondedto a carbon atom or to another element.

The term “nitro” refers to the —NO₂ group.

The term “thio” refers to a compound described previously herein whereina carbon or oxygen atom is replaced by a sulfur atom.

The term “sulfate” refers to the —SO₄ group.

The term thiohydroxyl or thiol, as used herein, refers to a group of theformula —SH.

More particularly, the term “sulfide” refers to compound having a groupof the formula —SR.

The term “sulfone” refers to compound having a sulfonyl group —S(O₂)R.

The term “sulfoxide” refers to a compound having a sulfinyl group —S(O)R

The term ureido refers to a urea group of the formula —NH—CO—NH₂.

Throughout the specification and claims, a given chemical formula orname shall encompass all tautomers, congeners, and optical- andstereoisomers, as well as racemic mixtures where such isomers andmixtures exist.

Certain compounds of the present disclosure may possess asymmetriccarbon atoms (optical or chiral centers) or double bonds; theenantiomers, racemates, diastereomers, tautomers, geometric isomers,stereoisometric forms that may be defined, in terms of absolutestereochemistry, as (R)- or (S)- or, as D- or L- for amino acids, andindividual isomers are encompassed within the scope of the presentdisclosure. The compounds of the present disclosure do not include thosewhich are known in art to be too unstable to synthesize and/or isolate.The present disclosure is meant to include compounds in racemic,scalemic, and optically pure forms. Optically active (R)- and (S)-, orD- and L-isomers may be prepared using chiral synthons or chiralreagents, or resolved using conventional techniques. When the compoundsdescribed herein contain olefenic bonds or other centers of geometricasymmetry, and unless specified otherwise, it is intended that thecompounds include both E and Z geometric isomers.

Unless otherwise stated, structures depicted herein are also meant toinclude all stereochemical forms of the structure; i.e., the R and Sconfigurations for each asymmetric center. Therefore, singlestereochemical isomers as well as enantiomeric and diastereomericmixtures of the present compounds are within the scope of thedisclosure.

It will be apparent to one skilled in the art that certain compounds ofthis disclosure may exist in tautomeric forms, all such tautomeric formsof the compounds being within the scope of the disclosure. The term“tautomer,” as used herein, refers to one of two or more structuralisomers which exist in equilibrium and which are readily converted fromone isomeric form to another.

Unless otherwise stated, structures depicted herein are also meant toinclude compounds which differ only in the presence of one or moreisotopically enriched atoms. For example, compounds having the presentstructures with the replacement of a hydrogen by a deuterium or tritium,or the replacement of a carbon by ¹³C- or 14C-enriched carbon are withinthe scope of this disclosure.

The compounds of the present disclosure may also contain unnaturalproportions of atomic isotopes at one or more of atoms that constitutesuch compounds. For example, the compounds may be radiolabeled withradioactive isotopes, such as for example tritium (³H), iodine-125(¹²⁵T) or carbon-14 (¹⁴C). All isotopic variations of the compounds ofthe present disclosure, whether radioactive or not, are encompassedwithin the scope of the present disclosure.

The compounds of the present disclosure may exist as salts. The presentdisclosure includes such salts. Examples of applicable salt formsinclude hydrochlorides, hydrobromides, sulfates, methanesulfonates,nitrates, maleates, acetates, citrates, fumarates, tartrates (e.g.(+)-tartrates, (−)-tartrates or mixtures thereof including racemicmixtures, succinates, benzoates and salts with amino acids, such asglutamic acid. These salts may be prepared by methods known to thoseskilled in art. Also included are base addition salts, such as sodium,potassium, calcium, ammonium, organic amino, or magnesium salt, or asimilar salt. When compounds of the present disclosure containrelatively basic functionalities, acid addition salts can be obtained bycontacting the neutral form of such compounds with a sufficient amountof the desired acid, either neat or in a suitable inert solvent or byion exchange. Examples of acceptable acid addition salts include thosederived from inorganic acids like hydrochloric, hydrobromic, nitric,carbonic, monohydrogencarbonic, phosphoric, monohydrogenphosphoric,dihydrogenphosphoric, sulfuric, monohydrogensulfuric, hydriodic, orphosphorous acids and the like, as well as the salts derived organicacids like acetic, propionic, isobutyric, maleic, malonic, benzoic,succinic, suberic, fumaric, lactic, mandelic, phthalic, benzenesulfonic,p-tolylsulfonic, citric, tartaric, methanesulfonic, and the like. Alsoincluded are salts of amino acids, such as arginate and the like, andsalts of organic acids like glucuronic or galactunoric acids and thelike. Certain specific compounds of the present disclosure contain bothbasic and acidic functionalities that allow the compounds to beconverted into either base or acid addition salts.

The neutral forms of the compounds may be regenerated by contacting thesalt with a base or acid and isolating the parent compound in theconventional manner. The parent form of the compound differs from thevarious salt forms in certain physical properties, such as solubility inpolar solvents.

Certain compounds of the present disclosure can exist in unsolvatedforms as well as solvated forms, including hydrated forms. In general,the solvated forms are equivalent to unsolvated forms and areencompassed within the scope of the present disclosure. Certaincompounds of the present disclosure may exist in multiple crystalline oramorphous forms. In general, all physical forms are equivalent for theuses contemplated by the present disclosure and are intended to bewithin the scope of the present disclosure.

In addition to salt forms, the present disclosure provides compounds,which are in a prodrug form. Prodrugs of the compounds described hereinare those compounds that readily undergo chemical changes underphysiological conditions to provide the compounds of the presentdisclosure. Additionally, prodrugs can be converted to the compounds ofthe present disclosure by chemical or biochemical methods in an ex vivoenvironment. For example, prodrugs can be slowly converted to thecompounds of the present disclosure when placed in a transdermal patchreservoir with a suitable enzyme or chemical reagent.

Following long-standing patent law convention, the terms “a,” “an,” and“the” refer to “one or more” when used in this application, includingthe claims. Thus, for example, reference to “a subject” includes aplurality of subjects, unless the context clearly is to the contrary(e.g., a plurality of subjects), and so forth.

Throughout this specification and the claims, the terms “comprise,”“comprises,” and “comprising” are used in a non-exclusive sense, exceptwhere the context requires otherwise. Likewise, the term “include” andits grammatical variants are intended to be non-limiting, such thatrecitation of items in a list is not to the exclusion of other likeitems that can be substituted or added to the listed items.

For the purposes of this specification and appended claims, unlessotherwise indicated, all numbers expressing amounts, sizes, dimensions,proportions, shapes, formulations, parameters, percentages, quantities,characteristics, and other numerical values used in the specificationand claims, are to be understood as being modified in all instances bythe term “about” even though the term “about” may not expressly appearwith the value, amount or range. Accordingly, unless indicated to thecontrary, the numerical parameters set forth in the followingspecification and attached claims are not and need not be exact, but maybe approximate and/or larger or smaller as desired, reflectingtolerances, conversion factors, rounding off, measurement error and thelike, and other factors known to those of skill in the art depending onthe desired properties sought to be obtained by the presently disclosedsubject matter. For example, the term “about,” when referring to a valuecan be meant to encompass variations of, in some embodiments, ±100% insome embodiments ±50%, in some embodiments ±20%, in some embodiments±10%, in some embodiments ±5%, in some embodiments ±1%, in someembodiments ±0.5%, and in some embodiments ±0.1% from the specifiedamount, as such variations are appropriate to perform the disclosedmethods or employ the disclosed compositions.

Further, the term “about” when used in connection with one or morenumbers or numerical ranges, should be understood to refer to all suchnumbers, including all numbers in a range and modifies that range byextending the boundaries above and below the numerical values set forth.The recitation of numerical ranges by endpoints includes all numbers,e.g., whole integers, including fractions thereof, subsumed within thatrange (for example, the recitation of 1 to 5 includes 1, 2, 3, 4, and 5,as well as fractions thereof, e.g., 1.5, 2.25, 3.75, 4.1, and the like)and any range within that range.

EXAMPLES

The following Examples have been included to provide guidance to one ofordinary skill in the art for practicing representative embodiments ofthe presently disclosed subject matter. In light of the presentdisclosure and the general level of skill in the art, those of skill canappreciate that the following Examples are intended to be exemplary onlyand that numerous changes, modifications, and alterations can beemployed without departing from the scope of the presently disclosedsubject matter. The synthetic descriptions and specific examples thatfollow are only intended for the purposes of illustration, and are notto be construed as limiting in any manner to make compounds of thedisclosure by other methods.

Example 1 Treatment of Cognitive Deficits Materials and Methods

Mice: Mice were kept in accordance with guidelines of the Johns HopkinsUniversity Institutional Animal Care and Use Committee. Six week oldmale C57BL mice were used for the experiments.

Prophylactic treatment with DON: Six week old C57B1 mice were treatedwith DON (1 mg/kg) in 0.5 mL saline or saline alone. 24 h after firstDON administration, groups of mice (n=8) were infected withEcoHIV/NDK-V5C5 (2×10⁶ pg p24/mouse) or treated with saline byintraperitoneal inoculation. A second dose of DON was administered 24 hpost-infection and then treatment was continued on alternate days untilthe end of the experiment (including behavioral testing). Thirty daysafter infection, all mice were subjected to behavioral testing in aradial arm water test measuring learning and memory. Treatments given tothe groups of mice were saline, saline+DON, HIV, and HIV+DON.

Therapeutic treatment with DON: The experimental design was for DONprophylaxis except that mice were infected first and DON administrationat 1 mg/kg every other day started on Day 26 after infection.

Radial water maze: A water tank (6 ft in diameter; 4 ft high) wasdivided into six swimming lanes using aluminum dividers. The tank wasfilled with water at room temperature up to the upper edges of swimminglane dividers. White washable Crayola paint for children (Hallmark) wasadded to make the water non-transparent. A submerged platform was placedrandomly at the end of one of the lanes, where it remains throughoutthat particular day, but was moved to another random location onsubsequent days, insuring that the mouse was using short term memory tolocate the platform on that day and not recalling where the platform wason previous days. Several three dimensional visual cues were placedaround the tank so the mouse could make an association between the cueand the location of the platform. The memory test was expected to last5-6 days using male C57B1 mice and 8-12 days using male 129×1 mice; itconsisted of five daily trials of 1 min; the trials started at the sametime each day. Each memory test was conducted in its entirety by asingle designated individual wearing consistently similar protectiveclothing to avoid distraction of animals. At the start of each trial,the mice were placed in the pool from a randomly chosen arm and thenumber of errors (entering an arm without the platform) was recordedover a 1 min period. After each error, the mouse was gently pulled backto the start arm for that trial. After each trial, if the mouse did notlocate the platform, the investigator guided the mouse to the platformand allowed the mouse to rest on the platform for 20 s. After fourtrials, the mice were returned to their home cages for 30 min and thenadministered a memory retention trial. The memory retention trialstarted in the same arm as trial 4 (the last training trial). A baselinewas considered to be established when control mice reached asymptoticperformance levels, that is one error or less on trials four and five.The scores for each mouse on the last three trial days were averaged andused for statistical analysis.

Plasma analysis: DON was spiked into untreated mouse plasma to generatestandards at concentrations from 10 nM to 100 μM at half-log intervals.Butanol with 3N HCl (250 μL) was added to standards and samples (50 μL)and centrifuged at 16,000×g for 5 minutes to precipitate proteins. Analiquot (200 μL) of the supernatant was incubated at 60° C. for 30minutes to derivatize DON into a more stable and lipophilic analyte aspreviously described (Alt et al., 2015). After derivatization thesamples were dried at 45° C. under a nitrogen stream. Samples wereresuspended in 50 μL 70/30 water/acetonitrile, vortexed and centrifugedat 16,000×g. Samples (2 μL) were injected and separated on an Agilent1290 equipped with a C18 column over a 5.5 minute gradient from 30-70%acetonitrile+0.1% formic acid and quantified on an Agilent 6520 QTOFmass spectrometer. Standards within the quantifiable range were used togenerate a standard curve. Samples below the limit of quantification(BLQ) were assigned a value of 0.

Brain analysis: Brain sections were weighed. DON was spiked intountreated mouse brain tissue to generate standards as described above.Butanol with 3N HCl (5 μL/mg) was added to standards and samples andhomogenized by pestle followed by centrifugation at 16,000×g. An aliquot(200 μL) of the supernatant was transferred to a new tube and processedas described in the plasma analysis section. Twenty microliters wasinjected for analysis as described hereinabove.

Glutamate CSF levels: CSF samples were derivatized with dabsyl chloridefor analysis. To prepare a dabsyl chloride stock, acetone was added todabsyl chloride to 10 mM concentration, vortexed, water bath sonicatedfor 10 minutes followed by heating to 60° C. for 10 minutes, vortexingagain, centrifugation for 5 minutes at 16,000×g and taking thesupernatant. Ten microliter standards of glutamate were prepared inartificial CFS by serial dilution. To each standard and sample, onevolume of 0.2 M sodium bicarbonate buffer (pH 9.0 containing 100 μMglutamate-d5 as internal standard) and 2 volumes dabsyl chloride stockwas added. Samples were incubated at 60° C. 10 minutes to derivatize.Samples (10 μL) were injected and separated on an Agilent 1290 equippedwith a SB-AQ column over a 4 minute gradient from 20-95%acetonitrile+0.1% formic acid and quantified on an Agilent 6520 QTOFmass spectrometer. Glutamate was quantifiable to 10 nM.

Statistical analysis: Prism software version 5.0 (GraphPad Software) wasused for statistical analyses, including unpaired Student's t-test,two-way analysis of variance (ANOVA) and log-rank analysis. A P valueless than 0.05 was considered statistically significant.

Results

Effect of DON on HIV infection and neuropathogenesis in mice: A smallanimal model of HIV pathogenesis was used in which conventionalimmunocompetent mice are infected with chimeric HIV, EcoHIV, whichcarries murine leukemia virus envelope protein gp80 in place of gp120 topermit infection of mouse cells and mice (Potash-2005). HIV-infectedmice seroconvert and mount protective CD8 T cell responses that limitvirus replication systemically and in brain, but can transmit infectiousvirus (Potash et al., 2005; Kelschenbach et al., 2012; Hadas et al.,2013). Chronically infected mice carry HIV in T cells, macrophages, andmicroglial cells in the brain (Kelschenbach et al., 2012; He e al.,2014) and similar to many HIV infected individuals, the animals manifestcognitive disease that can be quantified by measuring animal performancein behavioral tests including water maze and fear conditioning. Twoexperiment formats were used to test the effect of DON on HIV infectionin this system: DON prophylaxis and DON treatment.

Effect of compound 25 on reversing EcoHIV-induced increases in CSFglutamate concentration: Male C57BL6 mice were sham or EcoHIV-inoculated(4e6 pg p24, i.p.) then randomized to receive vehicle or compound 25(1.82 mg/kg, i.p. every other day) beginning 15 post-inoculation. On day33 post-inoculation, all mice were deeply anesthetized with isoflurane30 minutes post-last dose, and CSF was terminally sampled from thecisterna magna prior to measurement of glutamate concentrations byLC/MS-MS. EcoHIV infection caused increases in CSFS glutamate that werenormalized by compound 25 treatment. Excluding values >2SD from mean,n=4-5/group. Analysis by two-way ANOVA followed by post-hoc Fisher'sLSD, *p<0.05, ***p<0.001. Data are presented as the mean+SEM.

FIG. 1A, FIG. 1B, and FIG. 1C show that prophylactic treatment of micewith DON prevents development of HIV-induced cognitive impairment.

FIG. 2A, FIG. 2B, and FIG. 2C show that DON treatment of EcoHIV-infectedmice with demonstrable cognitive impairment completely abrogates(reverses) cognitive disease.

FIG. 3 shows that DON treatment reversed HIV-mediated down-modulation ofSTX1A and NRGN in correlation with abrogation of cognitive defect ofEcoHIV-infected mice.

FIG. 4A, FIG. 4B, and FIG. 4C show that micromolar DON levels wereobserved in plasma, CSF and basal ganglia in mice treated in thebehavioral experiments. Mouse samples were analyzed for DON usingLC-MS/MS 15 minutes post administration. DON was quantifiable in allsamples provided. Fifteen minutes after dosing, DON averaged around2.5-3 μM in plasma, and 0.3-0.5 uM in brain tissue and CSF. Nosignificant difference was observed between mice infected with PBS andEcoHIV.

FIG. 5A shows glutamate in CFS from mice treated with DON as quantifiedvia LC/MS. There was a trend for glutamate levels to be higher in EcoHIVmice versus PBS mice. In addition, DON tended to decrease the glutamatein the EcoHIV mice; however, no significant differences were found amongthe treatment groups by one-way ANOVA analysis.

FIG. 5B shows that compound 25 significantly reversed EcoHIV-inducedincreases in CSF glutamate concentration in the EcoHIV mice.

DISCUSSION

Using the prototype glutamine antagonist DON, it is shown for the firsttime, at doses less than that used for its anticancer efficacy, that DONcan selectively abrogate cognitive deficits, for example, cognitivedeficits in the EcoHIV model of neuroAIDs. The presently disclosedsubject matter demonstrates the use of glutamine antagonists (or theirprodrugs or analogs) for the treatment of cognitive deficits, such asasymptomatic neurocognitive impairment (ANI), mild neurocognitivedisorder (MND), and severe HIV-associated dementia (HAD).

Example 2

Exemplary DON prodrug reverses cognition deficits in the experimentalautoimmune encephalomyelitis (EAE) murine model of Multiple Sclerosis

Methods

Background; It is well known that EAE is an animal model of braininflammation, and in particular is the most commonly acceptedexperimental model for the human inflammatory demyelinating disease,multiple sclerosis. As shown in FIG. 6 , a previous study demonstratedthat DON administered i.p. q.a.d at a dose of 1.6 mg/kg from the time ofimmunization attenuates EAE (Shijie, et al., 2009), but lower doses wereineffective. No other studies on DON in EAE have been reported.

Prevention Study: C57BL/6 mice immunized with MOG 35-55 to induce EAE(Rahn, et al., 2012; Hollinger, et al., 2016). Mice administered Vehicleor an exemplary DON prodrug (1 mg/kg equivalent) p.o. q.d. from day ofimmunization, monitored for body weight and EAE disease score.

Treatment/Cognition Study: C57BL/6 mice immunized with MOG 35-55 toinduce EAE (Rahn, et al., 2012; Hollinger, et al., 2016). Miceadministered Vehicle or DON prodrug (1 mg/kq equivalent) p.o. q.a.d.from appearance of disease symptoms (EAE score ≥1). Mice monitored dailyfor body weight and EAE disease score. Mice tested for cognition in theBarnes maze (4 trials per day over 4 consecutive days) (Rahn, et al.,2012; Hollinger, et al., 2016) >8 wks after treatment began, whendisease/disability scores had normalized between groups so thatcognitive scores could be compared (P<0.99).

Summary: Similar to a past report using DON, we found that our exemplaryDON prodrug prevented EAE-induced clinical scores. Specifically, asshown in FIG. 7 , the DON prodrug completely prevents the development ofphysical signs of EAE. The magnitude of the therapeutic effect, however,was greater using our prodrug. We extended these findings to show thatour exemplary DON prodrug reduced EAE-induced clinical scores using atreatment paradigm in which the drug was not administered until theonset of physical signs of EAE (FIG. 8A and FIG. 8B). We also report forthe first time that the exemplary DON prodrug reverses EAE-inducedcognitive deficits as measured by performance in the Barnes maze (FIG. 9, FIG. 10A and FIG. 10B). These data suggest that DON prodrugs can beused to treat or prevent cognitive deficits in subjects suffering frommultiple sclerosis.

Example 3 DON Attenuates Cognitive Deficits Methods

Compounds: Lipopolysaccharide (LPS; 0111:B4, Lot 115M4090V) was obtainedfrom Sigma-Aldrich and dissolved in ice cold filtered phosphate bufferedsaline (PBS) on the day of administration. Compound 9 was synthesized byour laboratory and dissolved in 3% ethanol in HEPES-buffered saline (50mM) on the day of administration.

Animals: Studies were conducted according to protocol #MO16M27 approvedby the Animal Care and Use Committee at Johns Hopkins University. MaleC57BL/6 mice were obtained from Envigo (Envigo RMS Division,Indianapolis, Indiana) at 9 weeks old and maintained on a 12 hourlight-dark cycle with ad libitum access to food and water. Mice wereallowed three days to habituate to the facility prior to fearconditioning and testing.

Fear Conditioning: Fear conditioning and testing were performed insound-attenuating chambers equipped with a metal grid floor to delivercalibrated inescapable footshocks. Fear conditioning (Day 1) consistedof a 2 minute habituation period followed by three 20 second 80 dB 2 kHztones co-terminating in 0.4 mA footshocks separated by 1 minuteinterstimulus intervals. One day later (Day 2), each mouse wasrandomized into one of four treatment groups (PBS/Vehicle, PBS/9,LPS/Vehicle, LPS/9) and co-administered either LPS (0.3 mg/kg, i.p.) orfiltered PBS with either 9 (1.9 mg/kg, i.p.) or Vehicle. Four hourslater, each mouse was exposed to a reactivation session in which theywere placed back in the fear conditioning context for 15 minutes. Oneday later (Day 3), each mouse underwent a test for fear memoryreconsolidation by being placed again in the fear conditioning contextfor 10 minutes. For each session, time spent freezing was determinedusing automated motion detection software (CleverSys) and converted topercentages.

Data Analysis: Freezing percentages during the reconsolidation test weregrouped by treatment into 2 minute bins. Between group differences weredetermined by repeated measure two-way ANOVA and post hoc Dunnett's testwith significance defined as p<0.05.

Results

LPS administration at a dose (0.3 mg/kg, i.p.) known to increaseperipheral and brain markers of inflammation impaired thereconsolidation of contextual fear memory. Mice that receivedLPS/Vehicle exhibited reduced freezing during the reconsolidation test.This effect was attenuated by co-administration of 9 (1.9 mg/kg, i.p.)(effect of time [F(4,144)=8.49, p<0.0001]).

CONCLUSION

Similar to its reported effects on conditioned fear acquisition andconsolidation (Pugh, et al., 1998; Thomson, et al., 2005), the presentstudy demonstrates that acute peripheral administration of LPS alsoimpairs reconsolidation of fear memory. The cellular mechanisms thatsubserve reconsolidation have yet to be fully delineated, but early worksuggests that this process shares several of the same molecularmediators with initial consolidation and is likely dependent onfunctional glutamatergic signaling (Li, et al., 2013; McGaugh, 2000;Suzuki, et al., 2004). Peripheral LPS administration inducespro-inflammatory cytokine release in the brain that perturbsglutamatergic signaling through various mechanisms (Potter, et al.,2013; Lee, et al., 2015), ultimately impairing the maintenance ofglutamate-dependent memory. Co-administration of 9 with LPS may act viaone or both of these mechanisms to attenuate subsequent impairment ofmemory reconsolidation.

Example 4 Compound 14b Enhanced CSF Delivery of DON in Monkey Method

Compound: Compound 14b was dissolved in 50 mM HEPES buffered salinecontaining 5% ethanol and 5% tween on the date of administration.

Monkey: Monkey studies were conducted according to protocol (#PR15M298)approved by the Animal Care and Use Committee at Johns HopkinsUniversity. Two female pigtail monkeys (approximately 3.5 kg, non-drugnaive) were adjacently housed in stainless steel cages on a socialinteraction rack (contains 4 cages, each 32.5″ wide×28″ deep×32″ high)maintaining temperature of 64-84° F., humidity of 30-70% withalternating 14-10 hour light/dark cycle as per the USDA Animal WelfareAct (9 CFR, Parts 1, 2, and 3). Food was provided daily in amountsappropriate for the size and age of the animals and RO purified waterprovided ad libitum through an in-cage lixit valve. Food enrichment wasprovided Monday through Friday.

Treatment: Prior to drug administration, macaques were sedated withketamine given as an intramuscular injection prior to test articleadministration. Sedation was maintained through blood and cerebrospinalfluid (CSF) sample collections with ketamine at a starting rate of 15mg/kg with additional doses of 20-30 mg during the first hour. Atsubsequent time points ketamine was given at 10-15 mg/kg. DON (50 mMHEPES buffered saline) and compound 14b (50 mM HEPES buffered salinecontaining 5% ethanol and 5% tween) were administered (1.6 and 3.6 mg/kgequivalent dose of DON) to the animals at a dosing volume of 1 mL/kgintravenously. CSF sample (target of 50 μL) was obtained by percutaneouspuncture of the cisterna magna at 30 min post dose. Blood samples (1 mL)were collected at 15 min, 30 min, 1 h, 2 h, 4 h, and 6 h post dose bypercutaneous puncture of a peripheral vein. Samples were processed forplasma (centrifuged at a temperature of 4° C., at 3,000 g, for 10minutes). All samples were maintained chilled on ice throughoutprocessing. Samples were collected in microcentrifuge tubes, flashfrozen, and placed in a freezer set to maintain −80° C. until LC/MSanalysis.

Data Analysis: DON was extracted from samples (50 mg) with 250 μLmethanol containing glutamate-d5 (10 μM ISTD) by vortexing in lowretention tubes. Samples were centrifuged at 16,000 g for 5 minutes toprecipitate proteins. Supernatants (200 μL) were moved to new tubez anddried at 45° C. under vacuum for 1 hour. To each tube, 50 μL of 0.2 Msodium bicarbonate buffer (pH 9.0) and 100 μL of 10 mM dabsyl chloridein acetone was added. After vortexing, samples were incubated at 60° C.for 15 minutes to derivatize. Samples (2 μL) were injected and separatedon an Agilent 1290 equipped with an Agilent Eclipse plus C18 RRHD2.1×100 mm column over a 2.5 minute gradient from 20-95%acetonitrile+0.1% formic acid and quantified on an Agilent 6520 QTOFmass spectrometer. Calibration curves over the range of 0.005-17.1 μg/mLin plasma and CSF for DON were constructed from the peak area ratio ofthe analyte to the internal standard using linear regression with aweighting factor of 1/(nominal concentration). Correlation coefficientof greater than 0.99 was obtained in all analytical runs. The meanpredicted relative standard deviation for back calculated concentrationsof the standards and QC's for all analytes were within the range of 85to 115%, except for the lowest concentration which was within the rangeof 80 to 120% with an overall accuracy and precision of 6.7% and 6.6%respectively.

Results

The pharmacokinetics of DON and compound 14b in monkeys were evaluated.In pigtail macaques, i.v. administration of DON (1.6 mg/kg) and compound14b (3.6 mg/kg; 1.6 mg/kg DON equivalent) demonstrated significantlydifferent DON plasma profiles (FIG. 12A). DON administration providedhigh plasma exposures with AUC0-t of 42.7 nmol*h/mL. In contrast,compound 14b administration delivered ˜7 fold lower plasma exposure ofDON with AUC0-t of 5.71 nmol*h/mL. The opposite observation was seen inthe CSF where enhanced DON levels were observed after compound 14badministration. In the CSF at 30 min post dose, DON administrationresulted in 0.33 nmol/g DON while compound 14b delivered 1.43 nmol/gDON. When comparing plasma to CSF ratio at 30 min, compound 14bdemonstrated unexpected 10-fold enhancement of DON CSF delivery versusDON (FIG. 12B).

Example 5 Compounds 14b and 47 Enhanced CSF Delivery of DON in SwineMethod

Compound: Compound 47 was dissolved in a sterile saline containing 5%ethanol and 5% Tween 80 on the date of administration.

Swine: Swine studies were conducted under a protocol approved by theJohns Hopkins Animal Care and Use Committee. Adult, femaleGöttingen×Yucutan miniature swine (Massachusetts General Hospital, MA)were housed in Johns Hopkins University facilities accredited by theAssociation for Assessment and Accreditation of Laboratory Animal CareInternational in compliance with the Animal Welfare Act, Animal WelfareRegulations, and the Public Health Service Policy on the Humane Care andUse of Laboratory Animals. Animals were maintained on a 14-h light and10-h dark schedule, provided ad libitum water and a commercial miniswinediet (Teklad, Madison, WI) with environmental enrichment(fruit/vegetables) twice daily.

DON and Compounds 14b and 47 Treatment: Animals were individually housedwhile on study in order to monitor behavior and clinical healthfollowing drug administration. Whole blood for drug pharmacokineticevaluation was collected from a dual lumen central venous catheter (CVC)implanted in the external jugular vein prior to study initiation.Animals were anesthetized with a combination of ketamine hydrochloride(20-30 mg/kg, i.m.) and xylazine (2 mg/kg, i.m.), intubated, andmaintained under isoflurane (1-2%) inhalant anesthesia. A temporaryperipheral saphenous vein catheter was placed in the hind limb to allowfor anatomical separation of drug infusion and whole blood sampling viaCVC. DON and compounds 14b and 47 were dissolved in a sterile salinesolution containing 5% ethanol and 5% Tween 80 prior to i.v. infusionvia saphenous vein catheter over 1 hour (1 ml/min) for a final dose of1.6 mg/kg or molar equivalent administered at 1 ml/kg (n=1/dose). Bloodsamples (1 mL) were taken from CVC at predose, 5, 15, 30, 45, and 60min. Plasma was separated by low speed centrifugation at 3000 g for 10min at 4° C. CSF was obtained from the cistema magna using a 3.5 in×22gauge spinal needle (Becton Dickinson Health Care, Franklin Lakes, NewJersey, USA) at 60 min post-dose. All samples were flash frozen uponharvest and stored at −80 C until bioanalysis.

Data analysis: Quantitation of DON in plasma, CSF, and brain homogenateby LC-MS/MS was performed. Briefly, DON was extracted from plasma, CSF,and brain samples with methanol containing glutamate-d₅ (10 μM ISTD) byvortexing followed by centrifugation 16000 g for 5 min. Supernatantswere aliquoted and dried at 45° C. for under vacuum for 1 h. Sodiumbicarbonate buffer (0.2M, pH 9.0) and dabsyl chloride (10 mM) in acetonewere added to each tube, mixed, and incubated for 15 min at 60° C. toderivatize. Samples were then injected and separated on an Agilent 1290equipped with an Agilent Eclipse plus C18 RRHD 2.1×100 mm column over a2.5 min gradient from 20 to 95% acetonitrile+0.1% formic acid andquantified on an Agilent 6520 QTOF mass spectrometer. Peak area ratio ofthe analyte to the internal standard was plotted against a 14 standardcurve to yield DON concentrations for each sample.

Result

The pharmacokinetic of DON, compound 14b and compound 47 were evaluatedin swine. IV administration of compounds 14b and 47 (1.6 mg/kg DONequivalent dose) resulted in 3-5-fold lower DON plasma exposuresrelative to an equimolar dose of DON (FIG. 13A). Plasma AUC_(0-t) forDON and compounds 14b and 47 were 29.9, 8.00 and 5.70 nmol-h/mL,respectively. The opposite trend occurred in CSF, where compounds 14band 47 delivered substantially higher amounts of DON to the CSF (FIG.13B), resulting in unexpected increased CSF-to-plasma ratios (FIG. 13C).

Example 6 Compound 25 Ameliorates Social Avoidance Behavior Induced byChronic Social Defeat Stress (CSDS) Method

Compound: Compound 25 was synthesized by our laboratory. For allpharmacokinetic and efficacy studies, compound 25 was administered peroral (p.o.) at a dose of 1.82 mg/kg (1 mg/kg DON equivalent) in 50 mMHEPES-buffered saline.

Mice: Male 7- to 8-week-old C57BL/6J (C57) mice (25-30 g; JacksonLaboratory, Bar Harbor, ME) and 4- to 6-month-old CD-1 retired breeders(35-45 g; Charles River Laboratories, Wilmington, MA) were used for allexperiments. Mice were housed on a reversed 12-h light/dark cycle, andmaintained in a humidity- and temperature-controlled room with water andfood available ad libitum. CD-1 mice were singly housed except duringsocial defeats. C57 mice were group housed before starting CSDS andsingly housed after CSDS. Behavioral experiments were conducted duringthe dark cycle. All studies were conducted with approved protocols fromthe Johns Hopkins University Institutional Animal Care and Use Committeeand were in accordance with the NIH guidelines for the Care and Use ofLaboratory Animals.

Treatment and results: Mice were exposed to CSDS and subsequentlyadministered compound 25 or vehicle every other day for 12 days (FIG.14A). To examine the effect of compound 25 treatment on behavioralphenotypes induced by CSDS, we first tested the mice in thethree-chambered social approach test, where the social approach of amouse toward a stranger mouse trapped in a wire cage was measured.Control mice exhibited significant preference for exploring a strangermouse (stranger 1) relative to an empty cage, as measured by the totalamount of time spent in each chamber and time spent sniffing each cage(two-tailed Student's t test, p=0.0025, FIG. 14B; p=0.0007, FIG. 14C).Consistent with previous reports, CSDS resulted in a social avoidancephenotype (Anacker et al, 2016; Hodes et al, 2014; Wook Koo et al,2016); mice in the CSDS group did not show preference for exploring thestranger mouse relative to the empty cage (two-tailed Student's t test,p=0.1523, FIG. 14B; p=0.1189, FIG. 14C). Chronic treatment of Compound25 normalized observed social behaviors in mice subjected to CSDS(two-tailed Student's t test, p=0.0036, FIG. 14B; p=0.0035, FIG. 14C),while having no effect on sociability in control mice (two-tailedStudent's t test, p=0.0005, FIG. 14B; p=0.0058, FIG. 14C). These resultssuggest that compound 25 can rescue CSDS-induced sociability deficit.

Example 7 DON Prevented Cognitive Decline in the EcoHIV-Infected MouseModel of HAND Method

Treatment and results: Similar to HAND patients, mice inoculated withEcoHIV exhibited impaired spatial learning and memory as measured byradial arm water maze (RAWM) 30 days post-infection (FIG. 15A). EcoHIVinfection resulted in a significant increase in the number of errors(FIG. 15A) and latency to escape (FIG. 15B) onto a hidden platform inthe maze. DON treatment (1 mg/kg, i.p., q.o.d.) beginning one day priorto EcoHIV or sham inoculation, and continued throughout the infectionperiod and RAWM testing, fully normalized cognitive performance asmeasured by both errors (main effect of treatment [F(3,140)=261.8,p<0.0001], trial [F(4,140)=146.2, p<0.0001], interaction[F(12,140)=1.93, p=0.0355]) and latency to escape (main effect oftreatment [F(3,140)=37.00, p<0.0001], trial [F(4,140)=56.03, p<0.0001],interaction [F(12,140)=1.832, p=0.0484]). Neither EcoHIV infection norDON treatment affected latency to escape to a visible platform (FIG.15C; main effect of trial [F(3,112)=34.44, p <0.0001]) indicating noimpairment in visual or motor function. Additionally, no mice exhibitedovert signs of toxicity (i.e. diarrhea, weight loss). TheEcoHIV-infected mice exhibited measurable viral loads in the spleen andbrain (FIG. 15D). DON treatment actually caused an increase inperipheral viral load (τ(14)=2.58, p=0.022) likely due to the welldescribed 14-mediated inhibition of T cell activity/proliferation61,which is known to be required for endogenous suppression of EcoHIVreplication. Therefore, despite enabling a modest increase in EcoHIVreplication, DON still prevented cognitive decline, suggesting itsmechanism of action to be secondary to the infection itself. Thesefindings suggest that glutaminase inhibition through DON delivery to theCNS prior to or during HIV infection may prevent the development ofcognitive impairment in HAND patients.

Unexpectedly, we have discovered that DON prevented the spacial memorydeficits in an animal model of HAND. We have also shown that DONprodrugs provide enhanced CNS delivery. Therefore, DON prodrugs areuseful for the treating of HAND in individuals infected by HIV.

Example 8 Compound 25 Ameliorates Anhedonia-Like Behavior Induced byCSDS Method

Sucrose Preference Test: The sucrose preference test was performedaccording to a previously described protocol (Cao et al, 2013; Roybal etal, 2007). The mice were singly caged before the test. On day 1, theirnormal water bottles were replaced with two 50 ml tubes (bottle ‘A’ andbottle ‘B’) fitted with bottle stoppers containing two-balled sippertubes. The position of bottles A and B were switched daily to avoid aside bias, and the fluid consumed from each bottle was measured daily.During days 1 and 2, bottles A and B were filled with normal drinkingwater (W/W). During days 3 and 4, both bottles were filled with asolution of 1.5% sucrose dissolved in drinking water (S/S). On days 5-8,bottle A contained 1.5% sucrose, and bottle B contained drinking water(S/S). Sucrose preference on each day for each mouse was calculated as100%×(Vol A/(Vol A+Vol B)) and averaged across the days for a givencondition (W/W, S/S, or S/W). The average of total fluid consumption wasalso calculated as (Vol A+Vol B).

Treatment and results: Mice subjected to CSDS have repeatedly been shownto exhibit reduced sucrose preference, an effect associated withstress-induced anhedonia (Krishnan et al, 2007). To examine the effectof compound 25 treatment on anhedonia-like behavior, control and CSDSmice were subjected to an 8-day sucrose preference test. Among allgroups of mice, there were no differences of fluid consumption betweenbottles A and B when filled with either normal drinking water (W/W, days1 and 2) or 1.5% sucrose solution (S/S, days 3 and 4), suggesting thatthere was no side preference of solution consumption during test days1-4. On days 5-8, control mice showed significant preference for thesucrose solution when compared with water consumption (two-tailedStudent's t test, p<0.01). Three-way repeated measures ANOVA revealed aCSDS×drug treatment interaction on the sucrose preference on days 5-8(F(1,29)=5.873, p=0.022). In agreement with previous studies (Krishnanet al, 2007), post-hoc tests indicated that mice in the CSDS groupexhibited decreased sucrose solution consumption compared with those incontrols (p=0.0028, 0.0155, and 0.0132 for days 6-8, respectively), withno differences in total fluid intake, suggesting decreased sucrosepreference induced by CSDS. Treatment with compound 25 markedly restoredsucrose consumption in the CSDS group (p=0.0039, 0.0003, 0.0016, and0.0018 for days 5-8, respectively, FIG. 16 ), whereas no change wasobserved in controls (p=0.7687, 0.9933, 0.9866, and 0.9871 for days 5-8,respectively), suggesting that compound 25 can rescue the anhedonia-likebehavior phenotype induced by CSDS.

Example 9 Compound Synthesis and Characterization

Representative compounds of the disclosure can be prepared as describedin PCT/US2016/044767 (WO 2017/023774 A1), which is fully incorporated byreference herein.

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All publications, patent applications, patents, and other referencesmentioned in the specification are indicative of the level of thoseskilled in the art to which the presently disclosed subject matterpertains. All publications, patent applications, patents, and otherreferences (e.g., websites, databases, etc.) mentioned in thespecification are herein incorporated by reference in their entirety tothe same extent as if each individual publication, patent application,patent, and other reference was specifically and individually indicatedto be incorporated by reference. It will be understood that, although anumber of patent applications, patents, and other references arereferred to herein, such reference does not constitute an admission thatany of these documents forms part of the common general knowledge in theart. In case of a conflict between the specification and any of theincorporated references, the specification (including any amendmentsthereof, which may be based on an incorporated reference), shallcontrol. Standard art-accepted meanings of terms are used herein unlessindicated otherwise. Standard abbreviations for various terms are usedherein.

Although the foregoing subject matter has been described in some detailby way of illustration and example for purposes of clarity ofunderstanding, it will be understood by those skilled in the art thatcertain changes and modifications can be practiced within the scope ofthe appended claims.

That which is claimed:
 1. A method for treating a subject having acognitive deficit or psychiatric disorder, the method comprisingadministering to the subject a compound having formula (I):

or a pharmaceutically acceptable salt thereof, wherein: X is selectedfrom the group consisting of a bond, —O—, and —(CH₂)_(n)—, wherein n isan integer selected from the group consisting of 1, 2, 3, 4, 5, 6, 7,and 8; R₁ is selected from the group consisting of C₁₋₆ alkyl andsubstituted C₁₋₆ alkyl; R₂ is —C(═O)—O—(CR₃R₄)_(m)—O—C(═O)—R₁₀; R₂′ isselected from the group consisting of H, C₁-C₆ alkyl, and substitutedC₁-C₆ alkyl; each R₃ and R₄ is independently H, C₁-C₆ alkyl, substitutedC₁-C₆ alkyl, aryl, substituted aryl, —(CR₃R₄)_(m)—NR₅R₆, or

m is an integer selected from the group consisting of 1, 2, 3, 4, 5, 6,7, and 8; R₅ and R₆ is independently H or alkyl; and R₁₀ is selectedfrom the group consisting of alkyl, substituted alkyl, cycloalkyl,substituted cycloalkyl, monosaccharide, acylated monosaccharide, aryl,substituted aryl, heteroaryl, and substituted heteroaryl, in an amounteffective to treat the cognitive deficit or psychiatric disorder.
 2. Themethod of claim 1, wherein X is —CH₂—.
 3. The method of claim 1, whereinX is —O—.
 4. The method of claim 1, wherein R₁ is selected from thegroup consisting of methyl, ethyl, isopropyl, cyclopentyl, cyclohexyl,trimethylammonium, triethylammonium, tri(hydroxyethyl)ammonium,tripropylammonium, and tri(hydroxypropyl)ammonium.
 5. The method ofclaim 1, wherein: m is 1; each R₃ and R₄ are independently H, C₁-C₆alkyl, aryl or substituted aryl; and R₁₀ is selected from the groupconsisting of alkyl, substituted alkyl, cycloalkyl, substitutedcycloalkyl, aryl, substituted aryl, heteroaryl, and substitutedheteroaryl.
 6. The method of claim 1, wherein the compound havingformula (I) is a compound having formula (II):

wherein: R₁ is selected from the group consisting of H and C₁₋₆ alkyl;R₃ and R₄ are independently selected from the group consisting of H,C₁-C₆ alkyl, substituted C₁-C₆ alkyl, aryl, and substituted aryl; andR₁₀ is C₁₋₆ alkyl.
 7. The method of claim 6, wherein R₁ is selected fromthe group consisting of methyl, ethyl, and isopropyl.
 8. The method ofclaim 6, wherein R₃ is H and R₄ is selected from the group consisting ofmethyl and phenyl.
 9. The method of claim 6, wherein R₁₀ is selectedfrom the group consisting of isopropyl and tert-butyl.
 10. The method ofclaim 1, wherein the compound having formula (I) is selected from thegroup consisting of:


11. The method of claim 10, wherein the compound having formula (I) isselected from the group consisting of:


12. The method of claim 1 for treating a subject having a cognitivedeficit.
 13. The method of claim 12, wherein the cognitive deficit ischaracterized by impairment of the mental processes of perception,learning, memory, judgment, and/or reasoning.
 14. The method of claim12, wherein the cognitive deficit is due to a viral infection.
 15. Themethod of claim 14, wherein the cognitive deficit is due to the humanimmunodeficiency virus (HIV).
 16. The method of claim 15, wherein thevirus is latent.
 17. The method of claim 12, wherein the cognitivedeficit is selected from the group consisting of asymptomaticneurocognitive impairment (ANI) and mild neurocognitive disorder (MND).18. The method of claim 12, wherein the cognitive deficit is associatedwith a neurodegenerative disorder.
 19. The method of claim 12, whereinthe cognitive deficit is associated with a psychiatric disorder.
 20. Themethod of claim 12, wherein the cognitive deficit is associated withmultiple sclerosis.
 21. The method of claim 1 for treating a subjecthaving a psychiatric disorder.